Obesity vaccines

Abstract

Obesity is one of the largest and fastest growing public health problems in the world. Last century social changes have set an obesogenic milieu that calls for micro and macro environment interventions for disease prevention, while treatment is mandatory for individuals already obese. The cornerstone of overweight and obesity treatment is diet and physical exercise. However, many patients find lifestyle modifications difficult to comply and prone to failure in the long-term; therefore many patients consider anti-obesity drugs an important adjuvant if not a better alternative to behavioral approach or obesity surgery. Since the pharmacological options for obesity treatment remain quite limited, this is an exciting research area, with new treatment targets and strategies on the horizon. This review discusses the development of innovative therapeutic agents, focusing in energy homeostasis regulation and the use of molecular vaccines, targeting hormones such as somatostatin, GIP and ghrelin, to reduce body weight.

Introduction

Obesity and overweight rates, from childhood to adulthood, have been increasing worldwide in the last decades and is now one of the most serious public health problems of the century.^1,2 Obesity is defined as a medical condition characterized by accumulation of excess body fat to the extent that it may have adverse effects on health.² The body mass index (BMI) is the measurement tool most routinely used in the clinic to diagnose overweight and obesity. BMI defines people as normal weight if between 18.5–24.9 kg/m², overweight when between 25 and 29.9 kg/m² and obese when greater than 30 kg/m². The main limitation of BMI is not providing information regarding body fat distribution, nonetheless, with few exceptions, correlates well with the percentage of body fat.^3,4

Obesity is a chronic disease with a high likelihood of weight regain after weight loss attained by medical therapies which requires a long-term approach.⁵ The adverse health consequences associated with weight gain start to increase at the upper limit of the normal BMI, from 22–24.9 kg/m², while obesity is a known risk factor for many chronic conditions, including type 2 diabetes mellitus, hypertension, metabolic syndrome, cardiovascular diseases, respiratory, musculoskeletal, infectious, psychiatric disorders and cancer,⁶ with a decrease in life expectancy to a similar extend as observed in smokers,⁷ as obesity negative effects on health are predicted to outweigh the positive gains derived from the declining in smoking rates.⁸ Since weight loss can largely improve or resolve most co-morbidities,⁹ obesity is currently considered the leading cause of preventable death worldwide.^4,10,11

Obesity is most often the result of a positive energy balance due to a combination of excessive food intake and lack of physical activity in genetically predisposed individuals, while only a limited number of cases are secondary to monogenetic causes, endocrine disorders or use of drugs that cause weight gain.⁶

Current obesity treatments

Diet and exercise are still the cornerstones for obesity treatment. Clinicians have few pharmacological tools for obesity management, while available anti-obesity drugs achieve only relative short-term weight loss and are often followed by weight regain.^3,5,12

Pharmacotherapy for obesity includes drugs that induce weight loss by suppressing appetite or altering nutrient absorption. These drugs are usually able to induce 5 to 10% weight loss when compared with placebo, the minimum requirement for a drug to be approved by the regulatory authorities such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA). In addition to weight reduction, in order to be approved for obesity treatment, drugs should prove to confer additional health benefits to the patient such as improvement of cardiovascular risk factors and demonstrate to have a good safety profile.¹³ Currently available anti-obesity drugs approved by the FDA include, appetite suppressants and the lipase inhibitor orlistat, of which only the former is authorized by EMEA and marketed in Europe. Appetite suppressant drugs include central nervous system stimulants, such as phentermine, a noradrenergic sympathetic amine approved for short-term treatment of obesity, which is the most widely used drug owing to its low cost and the fact of being marketed for decades. Phentermine commonly associated side effects include insomnia, irritability and increase in blood pressure.⁵ The FDA has more recently approved two new drugs to treat obesity, lorcaserin, a selective 5-hydroxytryptamine receptor 2c agonist¹⁴ and a fixed dose combination of phentermine with topiramate.¹⁵ Lorcaserin is indicated as an adjunct therapy to diet and exercise for chronic weight management of obese or overweight individuals with BMI over 27 kg/m² associated with hypertension, dyslipidemia or diabetes, however it has been associated with several safety concerns that include cardiac valvulopathy and increased risk of psychiatric, cognitive, and serotonergic adverse effects.¹⁶ Topiramate is a sulfamate-substituted monosaccharide marketed since 1996 and formerly approved for seizure disorders and prevention of migraine headaches, which was shown to promote weight loss as a side effect of their therapeutic usage in clinical trials. Previously to FDA approval, topiramate was used off-label as adjuvant therapy in obesity, along with the anti-depressives, fluoxetine, sertraline and buproprion, and the anti-diabetic drug, metformin.¹² Orlistat is a lipase inhibitor that prevents hydrolysis of dietary triglycerides and consequently prevents the absorption of dietary fat that is excreted unaltered by the gastro-intestinal tract. Orlistat is the only drug approved for long-term use, however it produces only a modest weight loss and is associated with high rates of gastrointestinal side effects, such as steatorrea, faecal incontinence and flatulence.¹⁷

For patients with BMI over 40 kg/m² or over 35 kg/m² associated with high-risk co-morbid conditions, in which previous medical weight loss treatments have failed, obesity surgery is the only therapy that proved to provide significant and sustainable weight reduction¹⁸ and is recognized by the National Institutes of Health.^19,20 In severe obesity, bariatric surgical treatments, have shown to be cost-effective at producing and maintaining weight loss, since decrease the risk of development of new obesity associated co-morbidities and to improve the existing ones.²¹ Patients submitted to bariatric surgery display a significant reduction in health care use rates and total direct health care costs, as well as a significant decrease in overall 5-y mortality rate, with a reduction in the relative risk of death of 89%.⁹ Understandably, in response to the relative ineffectiveness of medical therapy and in spite of the potential risks associated with an invasive procedure, the demand for bariatric surgery has been increasing.²²

Regulation of food intake and energy homeostasis

Energy homeostasis is highly regulated by physiological systems located in several brain centers, such as the hypothalamus, brainstem and reward centers in the limbic system, which control food intake and energy expenditure. In the basal hypothalamus are located two important food intake-regulating nuclei, the arcuate (ARC) and the paraventricular nucleus (PVN). The ARC receives signals from the periphery and plays an integrative role in appetite regulation, while projects second order neurons to the PVN, which is involved in the regulation of visceral efferent activity. In the ARC, there are two well characterized neuronal populations, one involved in stimulating appetite that co-expresses neuropeptide Y and Agouti related protein (NPY/AgRP) and another involved in inhibiting appetite that co-expresses proopiomelanocortin and cocaine and amphetamine regulated transcript (POMC/CART).²³ NPY is the most potent signal in the central nervous system that stimulates food intake and decreases energy expenditure, while POMC is a precursor protein that through proteolytic cleavage originates various peptides, including α -melanocyte-stimulating hormone (α-MSH) that decreases appetite and increases energy expenditure.^24,25 These centers are modulated by neural and hormonal signals coming from the periphery, namely hormones synthesized by the adipose tissue, such as leptin that reflect the long-term nutritional status of the body and is able to influence body weight regulation, and gastro-intestinal hormones, such as ghrelin, peptide tyrosine-tyrosine (PYY) and glucagon-like peptide 1 (GLP-1) that acutely modulate these pathways and are able to regulate food intake and energy expenditure.²⁶

Ghrelin

Ghrelin is a gastro-intestinal hormone that promotes food intake, decreases resting energy expenditure²⁷ and decreases spontaneous locomotor activity²⁸ leading to increased body weight mostly due to accumulation of fat.^29-31 Ghrelin is produced predominantly in the gastric fundus³² and conveys orexigenic signals to the hypothalamus.²⁹ Ghrelin acts in the ARC of the basal hypothalamus, stimulating the production and release of NPY and suppressing POMC.³³

Ghrelin has a unique posttranslational acylation at its 3-serine residue, mediated by the enzyme ghrelin-O-acyltransferase (GOAT), essential for the endocrine actions of the hormone, since acyl-ghrelin that accounts for 10% of circulating ghrelin, is the form which activates the growth hormone secretagogue receptor type 1a (GHS-R1a) to stimulate food intake and growth hormone secretion³² in contrast to des-acyl ghrelin which fails to activate the receptor.³⁴

Ghrelin plasma levels rise before meals and are suppressed after food intake³⁵ in lean but not in obese patients.³⁶ In most patients there is a negative correlation between fasting ghrelin levels and body mass index, reflecting the presence of an adequate physiological response, with low or high ghrelin levels in the context of positive or negative energy balance, respectively. Consequently, fasting ghrelin levels are usually low in obese subjects compared with controls,³⁷ although levels rise after diet induced weight loss.^35,38 In contrast, patients with nervous anorexia have high fasting ghrelin levels.³⁹ Therefore, with the exception of patients with Prader-Willi syndrome that display high circulating ghrelin levels that is thought to drive their hyperphagia and obesity⁴⁰ and rare mutations in the ghrelin/preproghrelin gene tenuously linked to early onset obesity,⁴¹ ghrelin does not seem to play a causative role in obesity and its decreased concentrations are believed to represent a physiological adaptation to the positive energy balance.

Ghrelin was also shown to mediate diabetic hyperphagia⁴² and to regulate glucose homeostasis by direct action in the pancreas, being able to supress insulin secretion in vitro and increase glucose in vivo,^43,44 while insulin reduces ghrelin levels suggesting the existence of an inverse relationship between the two hormones.⁴⁵

In addition, bariatric surgery has been demonstrated to interfere with appetite regulating pathways, inducing changes in gastro-intestinal hormones levels that could contribute to weight loss, in particular by suppressing the rise in ghrelin levels that is normally observed after caloric deprivation.^35,46

Vaccination Targets for Obesity Treatment

Adipose tissue antigens

Obesity has been shown to be associated with a chronic inflammatory response, therefore a vaccination approach toward auto-antigens to induce immune tolerance has been hypothesized and attempted. Oral immunization against pooled antigens derived from adipose tissue was used to modulate the inflammatory response. This therapeutical strategy demonstrated be safe and able to cause a significant reduction in waist and tight circumferences as well as improvement of lipid profiles, with a decrease in triglycerides and increase in HDL-cholesterol levels, despite having only a negligible effect on body weight⁴⁷ (Table 1). This obesity vaccine is an attractive one, due to its simplicity and lack of negative effects on metabolism or health, however it lacks specificity since the pooled adipose tissue antigens have not been fully characterized and could benefit from a more focused approach after identification of the more appropriate and effective molecular targets.

Table 1. Anti-obesity vaccine antigens targets, immunogenic approaches and results.

Target antigen	Vaccine principle	Type of vaccine	Vaccination results	Species/Citation
Adipose tissue antigens	To decrease inflammatory response by inducing immune tolerance toward self-antigens	Oral immunization against adipose tissue antigens	Reduces waist and tight circumferences. Decreases triglycerides and increases HDL-cholesterol. Negligible effect on body weight.	Human 47
Somatostatin	To prevent inhibitory effects of somatostatin, in order to increase the endogenous levels of GH and IGF-1	Chimeric-somatostatin	No change in food intake. 10% decrease body weight gain under high fat diet.	Mice 49
Glucose-dependent insulinotropic polypeptide (GIP)	GIP blockade to increase fat oxidation and improve insulin resistance.	Immunoconjugate of GIP covalently attached to the Qβ bacteriophage.	Protects against diet induced obesity. Decreases fat accumulation. Increases resting energy expenditure. No changes in locomotor activity.	Mice 54
Ghrelin	To suppress endogenous ghrelin bioactivity	Ghrelin conjugated to BSA (plus adjuvants).	Decreases food intake by 15% Decreases body weight by 10%	Pigs 73
		Ghrelin haptens conjugated to KLH (plus adjuvants).	Decreases body weight gain and body fat. Decreases food efficiency.	Rats 74
		Chemical conjugate of active ghrelin with NS1 - BTV protein (no adjuvants)	Increases energy expenditure. Non-significant decrease in long-term food intake and weight gain. Decreases NPY gene expression in the basal hypothalamus.	Mice 80

Somatostatin

The obese state is also characterized by a decreased growth hormone (GH) basal secretion and GH administration has been shown to reduce adiposity and increase lean mass.⁴⁸ Somatostatin is a peptide hormone produced in the hypothalamus as well as in other tissues such as the gastro-intestinal system, which inhibits GH and insulin-like growth factor 1 (IGF-1) secretion. Somatostatin based immunization procedures where formulated with the aim of removing the physiological inhibitory effects of somatostatin on the somatotropic axis in order to increase the endogenous levels of GH and IGF-1, without the need of direct usage of these anabolic hormones. In diet induced obese mice, anti-somatostatin vaccination has shown not to interfere with food intake nevertheless allowed to decrease body weight gain by 10% despite continuous feeding with high fat diet⁴⁹ (Table 1). This vaccination approach, although not aimed at a direct regulatory pathway of energy homeostasis, has the benefit of having a specific molecular target, somatostatin, which is capable to up regulate several hormones, in particular GH that has advantageous metabolic effects and decreases fat mass. However, there is still a need to assess the possible shortcomings on glucose metabolism secondary to the induced GH excess and to evaluate the possible risk of promoting neuroendocrine cell proliferation that are usually inhibited by somatostatin.

Glucose-dependent insulinotropic polypeptide

Glucose-dependent insulinotropic polypeptide formerly known as gastric inhibitory peptide (GIP) is a gastro-intestinal hormone secreted by the intestinal K-cells in response to the ingestion of carbohydrate and fat that stimulates glucose-dependent insulin release and secretion.⁵⁰ Besides the pancreas, the GIP receptor (GIP-R) is widely distributed in peripheral organs including the adipose tissue where it holds a key role in fat deposition and lipid metabolism.⁵¹ In wild type animals, GIP-R knockout is associated with mild impairment of glucose tolerance and insulin secretion, conversely in mice exposed to high fat diet, GIP-R knockout results in decreased weight gain, decreased tissue triacylglyceride storage and increased uncoupling protein-1 in brown adipose tissue suggesting the existence of increased energy expenditure.⁵² Functional GIP blockade using receptor antagonists have also shown to prevent or reverse obesity in rodent models, as well as increase fat oxidation in the liver and muscle and improve insulin resistance.⁵³

A vaccination approach for GIP inhibition has also been attempted. To induce the production of anti-GIP neutralizing antibodies, an immunoconjugate of GIP covalently attached to the Qβ bacteriophage has been developed. Anti-GIP vaccine inoculation has been shown to protect against diet induced weight gain due to decreased fat accumulation and increased energy expenditure secondary to a rise in basal metabolic rate and without changes in locomotor activity, results which were similar to the effects produced by chemical blockade of the GIP receptor⁵⁴ (Table 1).

Despite GIP not having a role in food intake regulation, GIP blockade was shown to be able to deplete adipose deposits and improve insulin resistance, therefore it has been considered a potential drug target for obesity related diabetes treatment.⁵⁵ By antagonizing GIP, there is also the possibility of inducing a negative insulinotropic effect, due to GIP role as incretin hormone that promotes glucose mediated insulin secretion. Since GIP does not has a role in food intake regulation and hyperglycemia has been shown to induce pancreatic resistance to the insulinotropic effects of the GIP hormone, this anti-obesity vaccine is particularly targeted for the obese patient with type 2 diabetes.

Ghrelin

Since ghrelin is the only identified orexigenic hormone, it has been considered a most promising target for the development of new obesity treatments,⁵⁶ which gave origin to several different research approaches in order to achieve neutralization of ghrelin biologic effects in energy homeostasis. As a proof of this concept, the effect of ghrelin blockade has been assessed in several experimental models which included genetic deletion of ghrelin or ghrelin receptor, antagonists of ghrelin receptor and GOAT inhibition.

Genetic deletion of ghrelin or GHS-R1a, the receptor that mediates ghrelins’ stimulatory effects on GH release and appetite, failed to show any significant defect in GH levels, linear growth and food intake or body weight in adult mice.^57-59 Nevertheless, ghrelin-deficient (Ghrl−/−) mice showed a decreased feed efficiency and were protected from the rapid weight gain induced by early exposure to high-fat diet, presenting decreased adiposity, improved serum profile of glucose and lipids, increased energy expenditure and locomotor activity.⁶⁰ In addition, ghrelin knockout mice compared with wild type mice also presented a decrease in respiratory quotient when fed with high fat diet, suggesting that endogenous ghrelin plays a more prominent role in determining the type of metabolic substrate that is used for maintenance of energy balance, decreasing fat utilization, than in the regulation of food intake.⁵⁷

These data may suggest that ghrelin is not an essential orexigenic signal. However, genetic deletions have the limitation of being prone to the development of adaptive mechanisms in neuroendocrine pathways that may have compensated the congenital lack of ghrelin signaling. Furthermore, unintentional ablation of the obestatin gene, also encoded by the ghrelin gene, which was shown to supress food intake, might further mask the potential of ghrelin in energy homeostasis.⁶¹ Moreover, the ghrelin receptor has been demonstrated to maintain a high level of constitutive activity even in the absence of the endogenous ligand,⁶² and its physiological relevance has been revealed by patients harboring missense mutations of the GHS-R with loss of its constitutive activity that are characterized by short stature and obesity.⁶³

Ghrelin receptor antagonists

Ghrelin receptor antagonists have demonstrated to decrease food intake, body weight and improve glucose tolerance due to increased glucose-dependent insulin secretion,⁴³ thus confirming the potential of ghrelin blocking as a potential treatment target for obesity and type 2 diabetes. Two ghrelin receptor antagonists (YIL-870 and YIL 871), despite the low circulating ghrelin levels, were found to promote weight loss and improve glucose tolerance in diet induced obesity (DIO) mice, suggesting that there is still sufficient ghrelin tone to allow the GHS-R1a antagonists to have a therapeutic effect not only in obesity but also in diabetes.⁴³ Ghrelin antagonism has been found to be specifically beneficial in the context of obesity combined with estrogen deficiency, being able to decrease food intake and body weight and increase uncoupling protein 1 (UCP-1) in brown adipose tissue, suggesting enhanced energy dissipation, in a mouse model of postmenopausal obesity.⁶⁴ The fact that ghrelin receptor is characterized by a high degree of constitutive signaling activity, raises questions about the physiologic importance of ghrelin in receptor activity regulation and whether an unknown endogenous inverse agonist exists, suggesting that the development of inverse agonist compounds could represent alternative valuable pharmacological tools potentially more efficient than neutral antagonists.⁶²

A different approach of neutralizing ghrelin consist in using ribonucleic acid Spiegelmer (SPM), a non-natural nucleic acid with specific binding activity toward a given molecule, synthetized as a mirror image of the biological target, which was shown to decrease food intake, promote weight loss and decrease food efficiency (weight gain per kilocalorie ingested), an indirect evidence of increased energy expenditure.⁶⁵ Anti-ghrelin SPM does not cross the blood-brain barrier due to its charge and size, thus ghrelin neutralization is thought to occur mainly in the periphery, though direct action in the ARC cannot be excluded, since the circunventricular area of the median eminence is characterized by an incomplete blood-brain barrier.⁶⁶

Ghrelin O-acyltransferase inactivation

The inactivation of GOAT, the enzyme that activates ghrelin, has been proposed as an alternative tactic to block ghrelin activity,⁶⁷ since antibodies targeted to hydrolyze the octanoyl moiety of ghrelin to form des-acyl ghrelin, which has no biological activity, resulted in increased metabolic rate and suppressed re-feeding after food deprivation.⁶⁸ A peptide-based GOAT specific antagonist inhibitor, the bisubstrate analog GO-CoA-Tat, composed of three elements: ghrelin, octanoyl Co-A and a Tat sequence that allows the analog to penetrate within the cell cytoplasm where ghrelin acylation occurs, has also been recently developed. The GO-CoA-Tat was shown to decrease serum levels of acyl ghrelin and prevent body weight gain; additionally, also demonstrated to increase insulin secretion and improve glucose tolerance.⁶⁹

Passive immunization

After a first attempt to produce a polyclonal anti-ghrelin antibody that dose-dependently inhibited fast-induced feeding and supressed dark phase food intake after intracerebroventricular administration,⁷⁰ Lu et al., produced the anti-ghrelin antibody, so called 33A, which binds with high affinity to the active form of the molecule, acylated ghrelin but not to des-acyl ghrelin.⁷¹ The inoculation of this monoclonal anti-ghrelin antibody inhibited acute ghrelin-mediated orexigenic effects, but was unable to change long-term food intake in mice. The antibody demonstrated to prevent the increase in food intake induced by exogenous administration of acyl-ghrelin, showing that 33A specifically blocked the activity of acylated ghrelin, however was unable to prevent the increase in food intake after a fast-refeeding challenge that is mediated by endogenous ghrelin. Since this anti-ghrelin antibody was shown to increase the ratio of acylated to des-acylated ghrelin, compensatory mechanisms might have been activated to counteract the blockage of peripheral ghrelin which suggests that blocking the peripheral ghrelin pathway alone may not be sufficient in the long-term.⁷¹

More recently, a mixture of monoclonal antibodies targeting different ghrelin haptens was shown to be required to maintain increased energy expenditure during fasting and deprivation-induced food intake, as well as to reduce overall food intake upon re-feeding. Passive immunization with a single high-affinity monoclonal antibody that targeted specifically the ghrelin N-terminus (JG4) did not in alter energy expenditure, fuel substrate utilization, or food intake in fasted mice. In contrast, combining JG4 with a C-terminally directed anti-ghrelin antibody (JG3), was able to increase energy expenditure and maintain the utilization of carbohydrate as a fuel substrate in fasted mice. Moreover, a “triplet” mAb cocktail combination that included the additional C-terminally directed antibody (JG2) that had been comparably ineffective in conjunction with JG3 or JG4 alone, not only promoted increased energy expenditure, but also reduced deprivation-induced food intake. Thus, suggesting that an oligoclonal response is necessary to maintain an increased whole body energy expenditure during fasting and re-feeding over a 24-h period, as well as to reduce overall food intake after fasting.⁷²

The main limitations of passive immunization approaches include development of acquired tolerance and lack of long-term effectiveness, due to the reduced half-lives of the antibodies and need of periodic administration, as well as the possibility of activation of compensatory pathways of ghrelin production in common with the other methods used for ghrelin inactivation.

Active immunization

Anti-ghrelin vaccines were developed with the rationale of inducing an immune response to prevent the interaction of ghrelin with its receptor and suppress endogenous ghrelin bioactivity. The first ghrelin immunization attempts included vaccines which used bovine serum albumin (BSA)⁷³ and keyhole limpet hemocyanin (KLH)⁷⁴ as carrier proteins and immunogenic substances (Table).

Ghrelin conjugated to BSA was used to immunize 19 wk old pigs in the presence of Freund’s incomplete adjuvant and diethylaminoethyldextran (DEAE). Two boost immunizations occurred 20 and 40 d after the first immunization and 14 d after primary immunization anti-ghrelin antibodies could be found in plasma of vaccinated animals. This immunoconjugate was able to decrease voluntary food intake by 15% and by the end of the experiment vaccinated animals weighed 10% less than control animals. As ghrelin is a known secretagogue of growth hormone, there were concerns associated with the effects of vaccination on the somatotropic axis, nonetheless such evidence was not found as growth hormone levels actually increased in vaccinated animals that was attributed to the normal feedback mechanism in response to dietary restriction and decreased fat mass.⁷³ Zorrilla, et al., produced 3 immunoconjugates using different ghrelin haptens: Ghr1 with N-terminal amino acids 1–10 of ghrelin, Ghr2 with C-terminal amino acids 13–28, and Ghr3 with the full-length rat ghrelin sequence (1–28) that were conjugated to KLH. Age- and weight-matched rats received immunizations on experimental days 0, 21, 35, 56, and 84. The first three immunizations consisted of Ribi emulsion adjuvant containing Ghr1-KLH, Ghr2-KLH, Ghr3-KLH, or KLH, while the last two inoculations used alum as adjuvant. Immunized rats ate normally but, once anti-ghrelin antibodies titers increased, body weight gain decreased with a decreased circulating leptin levels and preferential reduction of body fat due to decreased feed efficiency, indicating a reduced energy thrift. The ratio of brain to serum ghrelin levels was also lower in rats with stronger anti-ghrelin immune responses.⁷⁴

In contrast, ghrelin vaccination using ghrelin-PADRE peptide, a synthetic Pan-DR helper T cell epitope which binds with high affinity to HLA-DR molecules being able to induce a high IgG immune response to various antigens coupled to PADRE, was able to induce the development of high anti-ghrelin antibody titers although it failed to show any effect on body weight or food intake.⁷⁵

The main limitation of these anti-ghrelin vaccination strategies are the need to use adjuvants in order to achieve an appropriate antibody response which may be associated with inflammatory responses, risk of exacerbated immune response or have limited use in humans.

Active immunization using virus-like particles

Virus Like Proteins (VLPs) consist of viral proteins that lack genetic material which prevents a possible reversion to a pathogenic phenotype, since these structures have a highly repetitive nature have the advantage of allowing B cell receptor cross-linking due to the ordered presentation of epitopes in the molecule surface that induce an efficient B cell activation with high immunogenicity regardless of the route of the immunization, also allowing the use of a small number of immunizations with lower quantity of vaccine.⁷⁶ When compared with classic immunization techniques, VLPs vaccines are generally more safe, efficient and cost-effective.⁷⁷ VLPs have been used as immunogenic molecules in several recombinant vaccines in the last few years in order to induce the production of specific antibodies against exogenous agents, such as hepatitis B and human papilloma viruses, as well as endogenous molecules with a preponderant role in chronic diseases,⁷⁸ such as, the anti-angiotensin vaccine developed for arterial hypertension treatment.⁷⁹

The VPL based anti-ghrelin vaccine consist of a chemical conjugate of active ghrelin with protein tubules of NS1 of the Bluetongue Virus (BTV). The main goal of this VLP based approach was to develop a safer and more effective anti-ghrelin vaccine that could be used for human treatment.⁸⁰ NS1 protein tubules, although not part of the viral capsid, possess the same immunogenic characteristics as classical VLPs^81,82. The choice of NS1 tubules as VLP-like carrier protein was driven by its previous use as a distribution system for molecules of prophylactic vaccines against common human infectious diseases, such as foot-and-mouth disease and influenza A virus^82,83.

The ability of the vaccine to trigger an immune response was tested in normal weight and DIO male mice that developed increasing titers of specific anti-ghrelin antibodies, confirming that a vaccine consisting of an immunoconjugate alone was able to trigger an immune response without the need adjuvants.⁸⁰ The antibodies titers attained after the immunization protocol were not very high, when compared with antibodies titers after common infectious diseases, which is also reassuring in safety concerns, since complete ghrelin neutralization as a strategy for obesity treatment is not desirable as ghrelin also intervenes in several key biological processes besides appetite regulation, such as growth hormone secretion, gastro-intestinal and neurological functions.⁵⁶

In accordance to the expected biological effects of ghrelin neutralization, vaccinated mice showed increased energy expenditure that usually translates into greater ease of weight loss and maintenance, as ghrelin is known to suppress energy metabolism by increasing the respiratory quotient through decreasing utilization of fat as energy.^29,57 Vaccinated animals showed a non-significant decrease in long-term food intake and weight gain, which could be attributed to the activation of compensatory mechanisms of energy homeostasis pathways. Since the regulatory mechanisms that control energy homeostasis and appetite are very complex processes and include highly redundant signaling pathways,³³ it is possible that the lack of significant differences in some biological parameters, such as food intake and body weight maybe due to activation of compensatory mechanisms for the decrease in available active ghrelin similar to what occurs in ghrelin knockouts.^57,59

There was also a significant decrease of NPY gene expression in the basal hypothalamus vaccinated DIO mice reflecting a decrease in central orexigenic signals.⁸⁴ In comparison, in vaccinated normal weight mice there were no significant differences in the genetic expression of NPY gene but the expression of POMC was significantly lower in the basal hypothalamus, the fact that these findings where only observed in normal weight mice, suggests the likelihood of a compensatory mechanism to the decreased orexigenic signals from the periphery in order to prevent a reduction in the feeding threshold of normal weight mice.⁸⁰

Anti-ghrelin vaccination was associated with a paradoxical increase in circulating ghrelin levels that did not appear to have biological effects in energy regulation and were proven to be due to the presence of circulating immune complexes of ghrelin-anti-ghrelin.⁸⁰ The rise in ghrelin levels after other vaccination strategies had been previously reported although the presence of circulating immunocomplexes was not documented.⁷⁵ The presence of circulating immunocomplexes that could be due to a lower elimination rate, has not been shown to suffer deposition in the kidney glomerular basement membrane or induce renal toxicity. As the anti-ghrelin vaccination did not interfere with ghrelin expression in the stomach, ghrelin appears to be normally synthetized in immunized animals and after neutralization of ghrelin biological activity does not seems to occur upregulation of ghrelin expression in order to maintain the homeostasis⁸⁰(Table 1).

Since ghrelin is a growth hormone secretagogue, ghrelin neutralization could induce alterations in GH/IGF-1 axis,⁸⁵ yet anti-ghrelin vaccination had no effect in GH and IGF-1 levels, suggesting the unlikelihood to cause endocrine adverse effects on the growth hormone axis. Furthermore, the anti-ghrelin vaccine was well tolerated with no signs of inflammatory reaction or toxicity.

Concluding Remarks

Obesity is one of major public health problems of the century for which pharmacological options remain quite limited. Unsurprisingly this is an exciting research area and several possible treatment targets have been recently identified. One of the most exciting strategies on the horizon is the possibility of targeting molecular factors associated with energy homeostasis regulation through the use of vaccines as a possible means to control a major global disease.

Ghrelin, being the only identified orexigenic hormone, has attracted particular attention with the development of different neutralization approaches in order develop alternative obesity treatments, which was shown to be an effective means of decreasing food intake and increasing energy expenditure, important contributions to establish a negative energy balance and promote weight loss. The available data also suggests that the utility of ghrelin antagonism as a broad anti-obesity agent is questionable. The role of ghrelin in food intake regulation seems to act predominantly in the adaptive behavioral response to conditions of low energy intake, driving hunger, rather than driving basal food intake or appetite, while most obese patients have low ghrelin levels, it is not expected for a vaccine to be effective in the absence of diet induced ghrelin rise. Obese patients that could benefit from these therapeutic approaches include patients with Prader-Willi syndrome that have high ghrelin levels, individuals enrolling a diet and exercise program as adjuvant therapy for weight loss and for the prevention of weight regain after weight loss.

Food intake is highly regulated by a complex neuroendocrine network with redundant pathways. Therefore, it is unlikely that inhibition of a single signaling pathway will have a significant impact on energy homeostasis. Antagonizing ghrelin pathway alone, or any of the other pathways explored so far as a means to interfere with body weight regulation, may not be effective for obesity treatment, while combination therapies to block multiple pathways will most likely be needed.

Glossary

Abbreviations:

AgRP

agouti related protein

ARC

arcuate nucleus

BMI

body mass index

BSA

bovine serum albumin

BTV

Bluetongue Virus

CART

Cocaine and amphetamine related transcript

DEAE

diethylaminoethyldextran

DIO

diet induced obesity

EMEA

European Medicines Agency

FDA

Food and Drug Administration

GH

growth hormone

GHS-R1a

secretagogue receptor type 1a

GIP

Glucose-dependent insulinotropic polypeptide

GIP-R

GIP receptor

GLP-1

glucagon-like peptide 1

GOAT

ghrelin-O-acyltransferase

IGF-1

insulin-like growth factor 1

KLH

keyhole limpet hemocyanin

α-MSH

α -melanocyte-stimulating hormone

NPY

neuropeptide Y

POMC

proopiomelanocortin

PVN

paraventricular nucleus

PYY

peptide tyrosine-tyrosine

SPM

ribonucleic acid Spiegelmer

UCP-1

uncoupling protein 1

VLPs

Virus Like Proteins

10.4161/hv.27537

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Sara Andrade for editorial assistance in the preparation of the manuscript. UMIB is funded by grants from FCT POCTI/FEDER Fcomp-01–0124-FEDER-015893 and Project grant EXPL/BIM-MET/0618/2012.