s2fieldsofstudy: Biology Medicine

text:
A Novel Glucagon-like Peptide-1 (GLP-1)/Glucagon Hybrid Peptide with Triple-acting Agonist Activity at Glucose-dependent Insulinotropic Polypeptide, GLP-1, and Glucagon Receptors and Therapeutic Potential in High Fat-fed Mice*

Background: Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon have important gluco-regulatory actions. Results: Fusion of amino acid sequences of GLP-1, GIP, and glucagon produces hybrid peptides with triple-acting agonist activity. Conclusion: Hybrid peptides possess beneficial biological actions equivalent, or superior to, activation of single receptors. Significance: Multitargeting peptides offer a new class of therapeutics for obesity and diabetes. Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA2]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA2]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA2]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA2]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA2]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA2]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA2]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.

Peripheral signals that control glucose homeostasis and energy regulation are carefully balanced and encompass a number of factors, including a variety of peptide hormones (1). The major focus on gut hormone-based therapies over the past decades has concentrated on single molecules that target one specific pathway (2). Although specific glucagon-like peptide-1 (GLP-1) 2 mimetics are used clinically for type 2 diabetes, the glycemic control and weight reductions achieved with certain types of gastric bypass surgery is markedly superior (3). Recognition is growing that these beneficial effects reflect changes in circulating levels of multiple peptide hormones that trigger a broad spectrum of pathways involved in glucose regulation and energy balance (4). Therefore, combining the activity of two or more regulatory hormones, with complementary biological actions, offers a favorable approach for the treatment of obesity and diabetes. In this context, GLP-1, glucose-dependent insulinotropic polypeptide (GIP), and glucagon possesses a number of biological effects that would suggest significant combined therapeutic effectiveness (2).
Together GIP and GLP-1 account for almost all of the well established physiological incretin effect and have powerful insulin-releasing and gluco-regulatory properties (5). Moreover, both peptides appear to have important pancreatic betacell protective actions and additional extra-pancreatic glucose lowering effects that further promote therapeutic applicability for diabetes (6). On the other hand, glucagon is classically regarded as an important hormone in maintaining normal glucose concentrations through enhanced hepatic glucose production (7). However, recent evidence now suggests that glucagon can be exploited therapeutically as a satiety factor, which also increases energy expenditure and body weight loss (8). thermore, transgenic mice overexpressing the glucagon receptor (R) in pancreatic beta-cells demonstrate increased insulin secretion and pancreatic beta-cell mass, with protection against impaired glucose tolerance following high fat feeding (9). Thus, it follows that design of a single hybrid peptide, capable of simultaneous activation of GLP-1, GIP, and glucagon Rs, would have substantially enhanced therapeutic promise for obesity and diabetes.
To generate just such a compound, we have constructed nine novel GLP-1/GIP/glucagon hybrid peptides. These hybrid peptides have been created through fusion of the key amino acid sequences of GLP-1, GIP, and glucagon known to be important for biological activity (see Table 1). Importantly, because all three peptides are substrates for dipetidyl peptidase IV (10,11), hybrid peptides with GIP or GLP-1-like N termini have substitution of the naturally occurring alanine L-isomer residue for a D-isomer, whereas peptides with a glucagon-like N terminus have substitution of alanine for serine at position 2. These specific modifications are known to impart DPP IV resistance and improve biological activity of respective parent peptides (11)(12)(13). We initially examined DPP IV resistance, in vitro insulin secretion, and in vivo glucose-lowering and insulinotropic actions of all hybrid peptides. The acute antidiabetic effects of the most efficacious hybrids were then evaluated in obese diabetic (ob/ob) mice. The most effective peptide, [dAla 2 ]GLP-1glucagon-GLP-1 ([dA 2 ]GLP/GcG), was progressed to a twice daily injection regime in high fat-fed mice to examine effects of chronic treatment on body weight, food intake, energy expenditure, nonfasting glucose and insulin, glucose tolerance, insulin sensitivity, locomotor activity, and aspects of metabolic rate. Finally, to elucidate potential mechanism of action, cAMP production capabilities of [dA 2 ]GLP/GcG were examined in cells transfected with either the GLP-1, GIP, or glucagon R, and in vivo gluco-regulatory and insulin-releasing activity was assessed in GIP, GLP-1, and double incretin R knock-out mice.

EXPERIMENTAL PROCEDURES
Peptides- Table 1 displays the amino acid sequence of the nine hybrid peptides used in this study, which were based on the structures of GLP-1, GIP, and glucagon. In addition, native GLP-1, GIP, and glucagon, along with [dAla 2 ]GIP, [dSer 2 ]glucagon, and [dAla 2 ]GLP-1 were used as control peptides. All peptides were purchased from GL Biochem Ltd. (Shanghai, China; greater than 90% purity). In addition to quality control data supplied with peptide purchased, all pep-tides were characterized in-house using MALDI-TOF MS, as described previously (14).
DPP-IV Degradation Assay-Peptides were incubated at 37°C in 50 mmol/liter TEA-HCl (pH 7.8; Sigma-Aldrich) with purified porcine DPP-IV (5 milliunits; Sigma-Aldrich) for 0, 2, 4, and 8 h. Degradation profiles were obtained using RP-HPLC analysis as described previously (14), and the HPLC peak area data were used to calculate the percentage of intact peptide remaining at time points during the incubation.
In Vitro Insulin Secretion-Effects of peptides on in vitro insulin secretion were examined using BRIN-BD11 cells whose characteristics have been reported previously (15). Briefly, BRIN-BD11 cells were seeded (150,000 cells/well) into 24-well plates (Nunc, Roskilde, Denmark) and allowed to attach overnight at 37°C. Following 40 min of preincubation (1.1 mmol/ liter glucose; 37°C), cells were incubated (20 min; 37°C) in the presence of 5.6 and 16.7 mmol/liter glucose with a range of peptide concentrations (10 Ϫ12 -10 Ϫ6 mol/liter). After 20 min of incubation, buffer was removed from each well, and aliquots (200 l) were stored at Ϫ20°C prior to determination of insulin by radioimmunoassay (16).
In Vitro cAMP Production-Effects of [DA 2 ]GLP-1/GcG, GLP-1, GIP, and glucagon on cAMP production were assessed in Chinese hamster lung cells transfected with either the human GIP-or GLP-1-R, as well as human embryonic kidney (HEK293) cells transfected with the human glucagon R (17). Cells were seeded (200,000 cells/well) into 96-well plates (Nunc) and washed with Hanks' balanced salt solution buffer before incubation with test peptides (10 Ϫ6 -10 Ϫ12 mol/liter) in the presence of 200 mol/liter 3-isobutyl-1-methylxanthine for 20 min at 37°C. After incubation, medium was removed, and the cells were lysed before measurement of cAMP using Parameter cAMP assay (R&D Systems, Abingdon, UK) according to the manufacturer's instructions.
Animals-Acute animal studies were carried out in male National Institutes of Health Swiss mice (Harlan Ltd., Blackthorne, UK; 12-14 weeks old), obese (ob/ob) mice (derived from the colony originally maintained at Aston University (18); 14 -16 weeks old), and also C57BL/6 mice with genetic deletion of either the GIP-or GLP-1 R and both incretin Rs (the background and generation of GIP, GLP-1, and double incretin R knock-out mice has been previously described (19)). Longer term experiments were performed in National Institutes of Health Swiss mice previously fed a high fat diet for 140 days    2 ]GLP/GcG, exendin-4 (both at 25 nmol/kg of body weight; intraperitoneal), or saline vehicle (0.9% (w/v) NaCl) were administered at 09:00 and 16:00 h over 21 days to high fat-fed mice. Food intake, body weight, nonfasting plasma glucose, and insulin concentrations were monitored at 2-4-day intervals. Oral and intraperitoneal glucose tolerance (both 18 mmol/kg of body weight) and insulin sensitivity (15 units/kg of body weight; intraperitoneal) tests were performed after 21 days of treatment. At the end of the treatment period, locomotor activity and aspects of metabolic rate were assessed over a 22-h period using Complete Laboratory Animal Monitoring System metabolic chambers (Columbus Instruments, Columbus, OH), as described previously (14).
Biochemical Analyses-Blood samples were collected from the cut tip on the tail vein of conscious mice into chilled fluoride/heparin glucose microcentrifuge tubes (Sarstedt, Numbrecht, Germany) at the time points indicated in the figures. Samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) for 1 min at 13,000 ϫ g. Plasma glucose was assayed by an automated glucose oxidase procedure using a Beckman glucose analyzer II (Beckman Instruments). Plasma insulin was assayed by a modified dextran-coated charcoal RIA (16).
Statistical Analysis-The results are expressed as means Ϯ S.E., and the data were compared using the unpaired Student's t test. Where appropriate, data were compared using repeated measures or one-way analysis of variance, followed by the Student-Newman-Keuls post hoc test. Incremental area under the curve (AUC) analyses for plasma glucose and insulin were calculated using GraphPad Prism version 5.0. Groups of data were considered to be significantly different if p Ͻ 0.05.  Table 2). In addition, all novel hybrid peptides were completely stable to the actions of DPP-IV up to and including 8-h incubations ( Table 2).

DPP-IV Stability-As
In Vitro Insulin Secretion- Table 2 displays the effects of all test peptides on insulin secretion at 5.6 and 16.7 mmol/liter glucose in BRIN-BD11 cells. At both glucose concentrations, all peptides (10 Ϫ6 mol/liter), with the exception of native glucagon at 16.7 mmol/liter glucose, significantly (p Ͻ 0.05 to p Ͻ 0.001) increased insulin secretion compared with respective glucose controls. All hybrid peptides displayed similar insulin secretory potencies (Table 2). Thus, none of the hybrid peptides displayed absolute superior insulinotropic actions when compared with the native or positive control peptides ( Table 2).
Acute Glucose-lowering and Insulinotropic Actions of Hybrid Peptides in Normal and ob/ob Mice-Native GIP and glucagon failed to elicit any significant insulin-releasing or glucose-modulating actions in normal mice at the dose employed when compared with glucose alone controls ( Table 2). In contrast, GLP-1 significantly reduced (p Ͻ 0.05) overall 0 -60 min AUC plasma glucose values and increased (p Ͻ 0.01) the overall insulin secretory response when compared with controls (  (Table 2). Consequently, the insulin-releasing and glucose-lowering capabilities of these three novel hybrid peptides were examined in ob/ob mice (  (Table 2). In ob/ob mice, [DS 2 ]GcG/ GIP exhibited only a mildly enhanced insulin secretory action when compared with glucose alone control (Fig. 1, a and b), whereas [DA 2 ]GIP/GLP-1 had no obvious beneficial effects (Fig. 1, e and f). However, [DA 2 ]GLP-1/GcG retained substantial and significant (p Ͻ 0.05 to p Ͻ 0.01) glucose-lowering and insulin-releasing actions in ob/ob mice (Fig. 1, c and d).
Persistent Glucose-lowering and Insulinotropic Actions of [DA 2 ]GLP-1/GcG in High Fat-fed Mice-When administered 4 ( Fig. 2, a and b) or 8 (Fig. 2, c and d) h prior to a glucose load [DA 2 ]GLP-1/GcG significantly reduced individual post-injection and overall 0 -60 min AUC glucose values in high fat-fed mice when compared with injection of native GLP-1 (Fig. 2, a  and c). In agreement, post-injection and overall glucose-induced insulin concentrations were markedly (p Ͻ 0.05 to p Ͻ 0.001) elevated by [DA 2 ]GLP-1/GcG administration 4 or 8 h prior to a glucose challenge when compared with native GLP-1 (Fig. 2, b and d).

Effects of Twice Daily Administration of [DA 2 ]GLP-1/GcG or Exenatide on Body Weight, Energy Intake, Nonfasting Plasma Glucose, and Insulin Concentrations in High Fat-fed Mice-
Twice daily administration of exenatide or [DA 2 ]GLP-1/GcG had no significant effect on accumulated energy intake over the course of the 21 days (Fig. 3a). There was an obvious trend for reduced body weight gain with [DA 2 ]GLP-1/GcG treatment, and this was significant on days 10 and 16 when compared with the saline control group (Fig. 3b). Similarly, nonfasting plasma glucose levels were not significantly different between groups at the individual observation points, but the overall glucose exposure during the 21-day period was significantly (p Ͻ 0.05) reduced by [DA 2 ]GLP-1/GcG treatment (Fig. 3c). Circulating insulin concentrations were significantly (p Ͻ 0.01) increased on day 21 in exenatide and [DA 2 ]GLP-1/GcG mice, with accompanying elevations (p Ͻ 0.05 and p Ͻ 0.01; respectively) of overall insulin levels during the entire 21-day treatment period (Fig. 3d).

Effects of Twice Daily Administration of [DA 2 ]GLP-1/GcG or Exenatide on Glucose Tolerance, Plasma Insulin Response to
Glucose, and Insulin Sensitivity in High Fat-fed Mice-Following intraperitoneal (Fig. 4, a and b) or oral (Fig. 4, c and d) glucose challenge on day 21, plasma glucose levels had a strong tendency to be reduced in exenatide and [DA 2 ]GLP-1/GcGtreated mice, but this failed to reach significance (Fig. 4, a and  c). Furthermore, there was a significant (p Ͻ 0.05 to p Ͻ 0.01) elevation of both post-injection and 0 -60 min overall AUC glucose-stimulated plasma insulin concentrations in exenatide and [DA 2 ]GLP-1/GcG mice following either intraperitoneal or oral glucose administration on day 21 (Fig. 4, b and d). The effects of [DA 2 ]GLP-1/GcG and exenatide were broadly similar (Fig. 4, a-d). In addition, the hypoglycemic action of exogenous insulin was substantially and similarly (p Ͻ 0.001) augmented in exenatide and [DA 2 ]GLP-1/GcG mice 30 and 60 min postinsulin injection on day 21 (Fig. 4e). This was corroborated from overall 0 -60 min glucose values, where both treatment groups significantly (p Ͻ 0.05) improved insulin action compared with saline controls (Fig. 4f).

Effects of Twice Daily Administration of Exenatide or [DA 2 ]GLP-1/GcG on Locomotor Activity and Metabolic Rate in
High Fat-fed Mice-There were no differences in O 2 consumption, CO 2 production, respiratory exchange ratio, and energy expenditure in any of the groups of mice on day 21 (data not shown). However, although there were also no significant differences in ambulatory activity between groups (Fig. 5, a and b), treatment with exenatide and [DA 2 ]GLP-1/GcG significantly (p Ͻ 0.05 and p Ͻ 0.01; respectively) increased rearing and jumping episodes during the light phase, as assessed by Z beam breaks (Fig. 5c). In addition, [DA 2 ]GLP-1/GcG administration significantly (p Ͻ 0.01) increased Z beam breaks during the dark phase (Fig. 5d).

Glucose-lowering and Insulinotropic Actions of [DA 2 ]GLP-1/ GcG in GLP-1, GIP, and Double Incretin R Knock-out Mice-As
would be expected, native GIP and GLP-1 were without biological effects in GIP and GLP-1 R knock-out mice, respectively (Fig. 6, a-d). In addition, administration of GIP or GLP-1 had no consequence in double incretin R knock-out mice (Fig. 6, e and f). In contrast, [DA 2 ]GLP-1/GcG significantly (p Ͻ 0.05 to p Ͻ 0.001) increased glucose-stimulated insulin secretion in GLP-1, GIP, and double incretin R knock-out mice (Fig. 6, b, d,  and f). The glucose-lowering actions of [DA 2 ]GLP-1/GcG were particularly evident in GIP R knock-out mice, with significant

DPP-IV stability, in vitro insulin secretory activity, and in vivo glucose lowering and insulin releasing actions of native, control, and novel hybrid peptides
Resistance of peptides to degradation by DPP-IV (5 milliunits) was measured (n ϭ 3) following 0, 2, 4, and 8 h of incubation. Reaction products were subsequently analyzed by HPLC. For in vitro insulin secretory studies, peptides (10 Ϫ6 M) were incubated with BRIN-BD11 cells in the presence of 5.6 or 16.7 mM glucose (20 min; n ϭ 8), and insulin release was measured by radioimmunoassay and presented as a percentage of respective control. For in vivo studies, plasma glucose and insulin concentrations were measured immediately prior to and 15, 30, and 60 min after intraperitoneal administration of glucose alone (18 mmol/kg of body weight; n ϭ 8) or in combination with respective peptides (each at 25 nmol/kg of body weight) in 18-h fasted NIH Swiss normal mice. The data are expressed as means Ϯ S.E.

GLP-1/Glucagon Hybrid Peptide
reductions in 30 and 60 min post-injection values (p Ͻ 0.001), as well as overall 0 -60 min AUC (p Ͻ 0.05) values, when compared with glucose controls (Fig. 6a). In addition, there was also a strong trend for decreased glucose levels with [DA 2 ]GLP-1/ GcG treatment in GLP-1R knock-out mice (Fig. 6c), and increased sample size may have improved the statistical power of this experiment. Interestingly, glucagon induced significant (p Ͻ 0.05 to p Ͻ 0.01) elevations of the overall glycemic excursion in GIP and double incretin R knock-out mice, but not in GLP-1 R knock-out mice (Fig. 6).

DISCUSSION
In the present study, we have evaluated the biological actions and therapeutic applicability of a series of novel GLP-1/GIP/ glucagon hybrid peptides. These peptides were engineered to combine the energy liberating action of glucagon (8), with the robust insulin-releasing actions of GIP and GLP-1 (6), in a single compound. Unlike native GIP, GLP-1, or glucagon, all novel peptides were completely stable to enzymatic breakdown by DPP-IV and exhibited significantly enhanced insulinotropic actions in clonal beta-cells. These observations are in harmony with previous studies that clearly reveal that the N-terminal modifications employed in the current study mask the DPP-IVbinding site and increase intrinsic biological activity of GIP, GLP-1, and glucagon (11)(12)(13). Thus, importantly our data show that the modified hybrid peptides still retain the ability to activate important corresponding pathways that lead to insulin secretion.
To screen acute in vivo properties of positive control and novel hybrid peptides, they were co-administered with glucose to normal mice. Only

GLP-1/Glucagon Hybrid Peptide
nificant reductions in body weight gain. Thus, it follows that combining the activity of two or more regulatory hormones, to concomitantly activate related biological pathways, offers a more favorable approach for the treatment of obesity and diabetes than activation of lone pathways (2). The slightly greater weight loss with [DA 2 ]GLP-1/GcG, as opposed to exenatide treatment, could also be an important factor. The observed effects of [DA 2 ]GLP-1/GcG were independent of changes in energy intake, despite related satiating effects of GLP-1 and glucagon (21,22). The lack of effect of exenatide on energy intake and body weight likely reflects up-regulation of inherent adaptive mechanisms to normalize energy balance and body weight regulation when only one signaling pathway is activated, with similar observations reported previously in our laboratory and others (23,24). However, this does contrast with similar studies in genetically obese or diabetic animals showing that longer term administration of exenatide significantly reduced food intake, causing weight loss (24 -26). The most plausible explanation for the lack of such chronic effects of exenatide in the current study therefore lies with the dose, possible GLP-1 receptor desensitization, or background genetics. It seems unlikely to us that the dose is an issue, because other studies with exenatide in animal models have employed doses of 1 nmol/kg (27), 24 nmol/kg (25), or 50 nmol/kg (24). The alter-  DECEMBER 6, 2013 • VOLUME 288 • NUMBER 49 native that GLP-1 receptor desensitization occurs is perhaps plausible from in vitro studies (28), although this phenomenon has not been observed to any appreciable extent in vivo (29,30). Thus, the observation of lack of effect of exenatide on energy intake and body weight regulation would appear to be speciesand animal model-specific.

GLP-1/Glucagon Hybrid Peptide
Intraperitoneal and oral glucose tolerance were marginally improved to a similar extent by 21-day twice daily treatment with [DA 2 ]GLP-1/GcG and exenatide. This was associated with significantly increased insulin levels following nutrient challenge. Thus, as would be expected, the metabolic benefits of [DA 2 ]GLP-1/GcG and exenatide are likely mediated predominantly by direct insulin secretory actions (21). Interestingly, substantial similar insulin-induced reductions of blood glucose levels were observed in [DA 2 ]GLP-1/GcG and exenatide mice, highlighting beneficial effects independent of pancreatic beta-cell function. This facet of biological action does not appear to be a direct consequence of reduced adipose tissue mass and thus most likely reflects actions of GLP-1 to improve insulin resistance (31). Thus, [DA 2 ]GLP-1/GcG appears to have both beneficial effects of pancreatic beta-cell function and also a direct or indirect augmentation of peripheral insulin action. We were unable to perform pharmacokinetic analysis of [DA 2 ]GLP-1/GcG because of the requirement for generation of a specific antibody. Thus, the possibility that [DA 2 ]GLP-1/ GcG has altered binding kinetics or an extended half-life as compared with exenatide cannot be discounted.
To further clarify the mechanism behind the observed effects of [DA 2 ]GLP-1/GcG or exenatide, we assessed aspects of locomotor activity and metabolic rate following 21-day treatment. Locomotor activity was unchanged, but explorative episodes (Z-beam breaks) were elevated during the light phase in both treatment groups, but only by [DA 2 ]GLP-1/GcG, and not exenatide, during the dark phase. This is interesting because the activity of mice is normally much less during the light phase and merits further investigation. Given the prominent effects of glucagon and GLP-1 on energy balance (32), the elevations of energy expenditure may have been predicted in the current study. However, this was not the case, because neither treatment regimen altered energy expenditure or the respiratory exchange ratio. In contrast, the beneficial metabolic actions of other co-agonists reported to date generally appear to center around effects on thermogenesis in brown adipose tissue and increased energy expenditure (2). However, it is unclear whether this effect would be fully translated to the obese insulin-resistant human form of type 2 diabetes. Thus, [DA 2 ]GLP-1/GcG may possess a distinct advantage over other similar coagonists and represent a particularly attractive candidate for further development.
Finally, in an attempt to delineate the receptors involved in the biological actions of [DA 2 ]GLP-1/GcG, we conducted studies in genetically transfected cells and knock-out mice. Interestingly, [DA 2 ]GLP-1/GcG stimulated cAMP production in GLP-1, GIP, and glucagon R-transfected cells with comparable, or even increased, efficacy when compared with the native peptide. Thus, our data clearly illustrate that [DA 2 ]GLP-1/GcG is a potent triple agonist, with cross-talk between GLP-1, GIP, and glucagon Rs. The rationale as to why a GLP-1/glucagon hybrid would efficiently activate GIP Rs is unclear, but it does demonstrate the marked similarities and sequence overlap between peptides and receptors of the same glucagon superfamily (33).
Moreover, recent data relating to a modified glucagon/GIP peptide hybrid clearly show that this molecule was capable of activating GLP-1 Rs (17). Further studies in GIP, GLP-1, and double incretin R knock-out mice confirmed our initial in vitro findings, with [DA 2 ]GLP-1/GcG displaying prominent insulin secretory actions in all three models, corroborating triple agonist properties. However, the glucose-lowering action of [DA 2 ]GLP-1/GcG was different in GIP and GLP-1 R knock-out mice, despite comparable insulin-releasing actions, indicating possible differences in insulin action between knock-out models.
In conclusion, the present study has demonstrated the hybrid peptide analog, [DA 2 ]GLP-1/GcG, is a DPP-IV resistant, potent, triple acting GIP, GLP-1, and glucagon R agonist.
GcG are due to concurrent activation of receptors on the same or distinct cell types, with subsequent stimulation of complementary signaling pathways, still needs to be determined. However, it is clear that multitargeting peptides are an attractive new class of therapeutics for the treatment of type 2 diabetes.


extfieldsofstudy: Biology Medicine

text:
Development of an in vitro bioassay for measuring susceptibility to macrocyclic lactone anthelmintics in Dirofilaria immitis☆

Graphical abstract

Introduction
The canine heartworm (Dirofilaria immitis) is an important filarial nematode parasite of dogs, cats, and other animals. The definitive host becomes infected through the bite of mosquitoes harboring infective third-stage larvae (L 3 ), which penetrate the bite wound and invade the subcutaneous tissues. Over a period of approximately 2 months, the larval stages of D. immitis migrate through the subcutaneous tissues and musculature before penetrating into the vasculature. Once in the vasculature, immature adult worms migrate to the pulmonary arteries and the right side of the heart, where they mature to adults (Kotani and Powers, 1982). Tissue-migrating larval stages are not associated with any recognizable pathology; however, once in the vasculature, worms can cause significant damage and life-threatening disease (Bowman and Atkins, 2009). Treatment of mature heartworm infection is costly and can cause serious adverse side effects, thus prevention by monthly administration of drugs that interrupt larval development is widely used (Bowman and Atkins, 2009). All compounds currently used for prevention of adult heartworm infection are members of the macrocyclic lactone (ML) drug class. In 1987, ivermectin became the first ML drug approved for use as a heartworm preventive (Campbell, 1989), and since then, products containing other ML drugs (milbemycin oxime, selamectin, and moxidectin) have been introduced. The US Food and Drug Administration (FDA) requires that all heartworm preventives achieve 100% efficacy when administered according to label directions (Hampshire, 2005). Therefore, when owner compliance with label directions is exact, no adult worms should develop in a dog administered one of these products. However, in recent years the FDA has received increasing reports of cases where mature worms develop in dogs despite evidence that monthly preventives were administered in a compliant fashion (Hampshire, 2005). These lack of efficacy (LOE) cases, are raising doubts that ML drugs achieve 100% protection (Geary et al., 2011). The two most obvious and common explanations for these LOE cases are (1) a lack of full compliance by the pet owners, which cannot be properly tested, or (2) that D. immitis has developed resistance to the ML drugs.
Resistance to the ML drugs is highly prevalent throughout the world in numerous species of gastrointestinal nematodes of livestock (Kaplan, 2004;Kaplan and Vidyashankar, 2012). Several in vitro bioassays have been developed and validated to detect ML resistance in many of these species (Taylor et al., 2002). One of these, the larval migration inhibition assay (LMIA) has already been successfully applied to several important species of nematode pathogens of sheep and cattle for diagnosis of ML resistance Kotze et al., 2006;Demeler et al., 2010) and appears particularly amenable for use with D. immitis.
Evidence suggests that ML drugs exert their anthelmintic effects on nematodes by binding to glutamate-gated chloride channels (GluCls) and hyperpolarizing motor neurons, resulting in paralysis and death (Wolstenholme and Rogers, 2005). This paralytic action of the ML drugs is the rationale behind the development of the LMIA, which measures the ability of larvae to migrate across a fine mesh sieve following incubation with increasing concentrations of drug. Motility of the infective L 3 of heartworms is critical for both the initial infection process, as well as for migration through the subcutaneous and muscular tissues of the canine host in order to gain access to the vasculature. Furthermore, the L 3 is one of the targets (along with the fourth-stage larvae) of monthly preventive treatment with ML drugs. Since L 3 can be produced in large numbers in properly equipped laboratories, this appears to be the ideal life cycle stage of D. immitis for use in an in vitro bioassay, and the LMIA appears to be the most suitable bioassay for quantifying the susceptibility of L 3 to the paralytic effects of ML drugs.
In this study, we optimized the LMIA for use with D. immitis L 3 . We then measured the ML susceptibility of a laboratory heartworm strain in the LMIA using both ivermectin and eprinomectin. This generated reproducible dose-response data, to which we applied a variable-slope nonlinear regression model, enabling us to calculate IC 50 values as a means of characterizing the in vitro susceptibility to these drugs. Validation of the LMIA for identifying reduced susceptibility in heartworm populations would provide an extremely valuable tool for monitoring the emergence and spread of anthelmintic resistance in D. immitis.

Parasites
An ML-susceptible strain of D. immitis (2005 Missouri strain) maintained and passaged in beagle dogs at the University of Georgia (Athens, GA) was used in this study. This strain was originally collected in 2000 from a dog at a Missouri animal pound, and while this donor dog was not treated with any MLs thereafter, prior treatment records are not available. Third-stage larvae were obtained by feeding microfilaremic blood to Aedes aegypti mosquitoes (black-eyed Liverpool strain) using artificial blood feeders, as previously described (McCall, 1981). Fourteen days after feeding, L 3 were obtained by gently crushing the infected mosquitoes, rinsing them onto a 32 lm mesh sieve set in either a Petri dish or a Baermann apparatus, and soaking them in warm Hanks' balanced salt solution. The Brugia pahangi L 3 (FR3 strain) used in some of the optimization experiments were obtained in the same manner, and are also maintained in beagle dogs at the University of Georgia.

Drug solutions
ML stock solutions (10 mM) were prepared by dissolving powdered ivermectin or eprinomectin (Sigma, St. Louis, MO) in DMSO (>99.5%; Sigma). Stock solutions were diluted with additional DMSO into 100Â working solutions ranging from 0.0312 to 2.0 mM. These were then added to culture media at a rate of 1% (v/v) to yield the concentrations used in the LMIA (0.312-20 lM in 1% DMSO). For each drug, the maximum concentration tested was restricted by its solubility in 1% DMSO.

DMSO concentration
The LMIA was performed with the Missouri strain of D. immitis using a 48 h incubation period and 2 h migration period. Fifty larvae per well were cultured in a 24-well plate in RPMI-1640 with antibiotics, as described above, with DMSO concentrations of 0%, 1%, and 2%. Assay results were scored as described for the optimized LMIA. Each concentration was tested in triplicate in three separate experiments.

Migration period
Migration tubes were prepared using a method modified from Wagland et al. (1992) and set in 400 ll RPMI-1640 plus antibiotics in the wells of a 48-well culture plate. A square of 20 lm mesh was secured over the bottom of each glass migration tube and held in place by a polymer collar fit around the tube. Tubes were suspended 2 mm above the bottom of the plate by a rubber o-ring fit around each collar. In a 100 ll volume, 50 B. pahangi L 3 were introduced to each of three migration wells. Larvae were allowed to migrate while incubating at 37°C and 5% CO 2 . For the first 3:45 h, the migration tubes were transferred to new wells in the plate every 15 min, and the tubes were finally removed 24 h after the beginning of the experiment. The number of L 3 to migrate over each time interval was then determined by counting the larvae in each well.

Incubation period
The LMIA was performed with the Missouri strain of D. immitis using either a 48 or 72 h incubation period and 2 h migration period. Fifty larvae per well were cultured in a 24-well plate in RPMI-1640 medium with 1% DMSO. Four independent experiments were performed, with each group tested in triplicate.

Mesh size
The LMIA was performed with the Missouri strain of D. immitis using a 48 h incubation period and 2 h migration period. Fifty larvae per well were cultured in a 24-well plate in RPMI-1640 medium with eprinomectin (0.625-50 lM) in 1% DMSO or in a DMSO-only control. Larvae were allowed to migrate through nylon mesh of either 25 or 30 lm pore size. Three independent experiments were performed, with each group tested in triplicate.
To determine the effect of larval diameter on migration, 25 D. immitis L 3 per well were cultured in a 24-well plate as above. Larvae that successfully migrated in the LMIA (n = 12) and those that failed to migrate (n = 12) were measured using an eyepiece micrometer on an inverted compound microscope (IX51; Olympus, Center Valley, PA) at 400Â magnification.

Optimized protocol for larval migration inhibition assay
After collection from mosquitoes, D. immitis L 3 were washed in RPMI-1640 and added to individual wells of a 24-well tissue culture plate (30 larvae per well) in 1 ml RPMI-1640 containing antibiotics and drug. Assay plates were then incubated at 37°C and 5% CO 2 for 48 h. Following this incubation period, acrylic migration tubes were placed into a new 24-well migration plate (Fig. 1); migration tubes were set in rows of six on a horizontal acrylic bar, as described by Demeler et al. (2010), and bottomed with 25 lm nylon mesh screens (Sefar, Inc., Heiden, Switzerland). With the migration tubes in place, 1.4 ml of culture medium warmed to 37°C was added over the tube in each well; drug concentrations in the media added to each well corresponded to those in the incubation plate. Using a dissecting microscope, all 30 larvae in each well of the incubation plate were collected and gently transferred in a volume of 200 ll to the corresponding migration tube. Once all the L 3 were transferred to migration tubes, they were placed back into the incubator at 37°C and 5% CO 2 for a 2 h migration period during which motile larvae may pass through the mesh screen (Supplementary video 1). After 2 h, the migration tubes were gently removed and the larvae that had migrated into each well of the migration plate were enumerated during examination with an inverted compound microscope. Three experiments were performed for each drug (ivermectin and eprinomectin) with concentrations tested in triplicate for each experiment.

Statistics
Differences in migration following incubation for 48 and 72 h (without drug) and differences in migration through meshes of different size were calculated using a one-tailed unpaired t-test. Correlation between larval diameter and the ability to migrate was calculated as a point-biserial correlation coefficient, with significance assessed by application of a two-tailed t-test.
Percent inhibition of migration was calculated for each well based on the number of larvae failing to migrate corrected for the percent of larvae that did not migrate in the no-drug control wells. Sigmoidal dose-response curves were calculated in Graph-Pad Prism Ò version 5 (GraphPad Software, Inc., San Diego, CA) using a variable slope nonlinear regression model. A four-parameter logistic equation was applied using global curve-fitting, with the bottom of the curves constrained to zero. For each treatment, the IC 50 and IC 95 (defined as the concentration effecting inhibition halfway between zero and the maximal response, and 95% of the maximal response, respectively) were calculated. R 2 values and 95% confidence intervals were calculated by GraphPad Prism Ò . Differences between the IC 50 values of IVM and EPR-treated groups were calculated using the extra sum-of-squares F test.

Migration period
B. pahangi, a filarioid parasite closely related to D. immitis was used in some preliminary experiments with the LMIA, since this parasite is easier to cycle and was readily available through the FR3. In these early experiments, we observed that almost all larvae that successfully migrated over 24 h did so within the first 2 h of incubation (mean ± SD = 99.2 ± 6.56%; Fig. 2). To ensure a convenient yet sufficiently long migration period, the migration rate of D. immitis L 3 over 1 h was determined, followed immediately by an additional 1 h in a separate migration plate. Significantly more larvae migrated over the combined 2 h (83%) period than 1 h (77%; p = 0.037), therefore a 2 h migration period was used for this LMIA.

Media and DMSO content
To determine the optimal culture medium for use in the D. immitis LMIA, RPMI-and NI-based media with added antibiotics were compared. Six groups of 50 L 3 were incubated in each medium with no difference observed in migration rates (p = 0.81; data not shown). The RPMI-based medium was chosen for future assays because of its ease of preparation and convenience.
To solubilize macrocyclic lactone anthelmintics in an aqueous media it is necessary to use DMSO, but this solvent is known to negatively affect nematode viability. To determine an appropriate solvent concentration, larvae were incubated in media containing 1%, 2%, or no DMSO and their migration rates assessed. While no decrease from control migration rates was observed in the 1% DMSO group (p = 0.39), a significant decrease was noted when larvae were exposed to 2% DMSO (p = 0.018; data not shown). Based on these data, 1% DMSO was used in the LMIA culture medium for all further experiments.

Incubation period
To allow sufficient exposure of the larvae to the drug without inducing an excessive reduction in migration of the untreated control larvae, it was necessary to optimize the incubation period. Comparison of the LMIA performed using 48 h or 72 h incubation periods showed that migration rates were significantly lower following the longer incubation (p = 0.011; Fig. 3). Furthermore, the logIC 50 for each assay was not significantly different (p = 0.51); therefore, a 48 h incubation period was selected for use in all future assays.

Larval diameter and mesh size
The pore size of the mesh sieve is a discriminating parameter for parasite migration in the LMIA. We observed that the diameter of L 3 varied somewhat, so we wanted to confirm that this variable would not affect migration rates in the LMIA. Larvae that had either migrated or failed to migrate through a 25 lm mesh were measured using an ocular micrometer in an inverted compound microscope (Fig. 4). The largest D. immitis L 3 diameter measured was 25 lm (mean ± SEM = 20.6 ± 0.268 lm). No correlation between migration and larval diameter was observed (p = 0.95), providing evidence that migration rates were not a function of variation in larval diameter.
Mesh size was selected so that it was no smaller than the largest larval diameter, yet remained fine enough to restrict the passage of immotile larvae. Given the commercial availability of mesh with a variety of pore sizes, migration rates through 25 and 30 lm mesh sieves were compared. No reduction in migration was observed for the smaller mesh size compared to the larger (three independent experiments, p = 0.38) for worms incubated without drug. However, migration of drug-treated larvae at the highest concentration tested was significantly reduced with the 25 lm mesh (p = 0.034; data not shown). A small percentage of larvae were observed to migrate through the 25 lm mesh at the highest drug concentrations tested, but it was confirmed visually that this was due to larval motility, and not the incidental passage of immotile larvae. In contrast, with the 30 lm mesh, small numbers of immotile larvae were found to pass through the mesh. Given these data, the 25 lm mesh size was selected as being optimal for use in this assay.

Optimized larval migration inhibition assay
The mean migration inhibition rates in the control wells were consistently less than 10% (mean ± SD = 7.78 ± 7.75%) in most assays, indicating that D. immitis L 3 retain high levels of motility after 48 h of incubation in RPMI media containing 1% DMSO. However, mean inhibition at the highest drug concentrations tested infrequently exceeded 90% (mean ± SD = 80.7 ± 13.9%). Dose-response curves were plotted for the ML-susceptible Missouri heartworm strain treated with either ivermectin or eprinomectin (Fig. 5) at concentrations ranging from 0.312 to 20 lM. The IC 50 values for D. immitis L 3 exposed to ivermectin and eprinomectin were 4.56 and 2.02 lM, respectively, and these values were significantly different from each other (p = 0.0428). The calculated IC 95 values were less reliable, characterized by very large 95% confidence intervals, especially for ivermectin (Table 1). The fit of each curve to the global nonlinear regression model was expressed as an R 2 value, and higher values were consistently found with eprinomectin, suggesting that eprinomectin is a better choice for use in this in vitro bioassay.

Discussion
The emergence of D. immitis that are resistant to macrocyclic lactone anthelmintics would represent a serious threat to canine health. However, there still is no consensus on the issue of whether ML resistance has emerged or if LOE cases are sufficiently   The diameters of larvae that successfully migrated in the LMIA and those that failed to migrate were measured. Horizontal line indicates mean, n = 12. explained by other factors. The major impediment to resolving this issue is the absence of a validated diagnostic test for measuring resistance in D. immitis. Here we report the development and optimization of an in vitro bioassay for measuring the in vitro susceptibility of D. immitis L 3 to ML anthelmintics. Using an established ML-susceptible laboratory isolate, we demonstrated the ability to reproducibly measure the in vitro dose-response characteristics of D. immitis L 3 to ivermectin and eprinomectin.
Considering the biology and epidemiology of D. immitis and its extreme sensitivity to ML drugs, the emergence of resistance in this parasite has historically been considered unlikely (Prichard, 2005). However, increasingly common LOE reports have raised concerns that ML resistance is developing in heartworms (Hampshire, 2005). In addition, several recent studies have reported genetic structural differences and/or sequence changes in some heartworm isolates leading these authors to conclude that resistance is present (Bourguinat et al., 2011a,b). Single nucleotide polymorphisms (SNPs) have been reported to occur in a gene encoding a P-glycoprotein, a class of proteins implicated in reduced ML efficacy against the filarioid Onchocerca volvulus (Prichard and Roulet, 2007), and associated with ML-resistance in Haemonchus contortus (Xu et al., 1998). Examining multiple D. immitis isolates, Bourguinat et al. (2011b) reported a correlation between the frequency of a pair of SNPs and reduced in vitro ML efficacy on microfilariae in an isolate-specific manner. In one case, a dog originating from Louisiana demonstrated persistent microfilaremia following multiple high-dose ML treatments (Bourguinat et al., 2011a). A high frequency of the aforementioned genotype was observed in this population of microfilariae, prompting the authors to cite ML resistance as the cause of this anomaly. However, even after numerous high-level doses of ML drugs, only 45% of the recovered microfilariae carried the SNPs suspected to be associated with resistance. Therefore, the significance of these SNPs as markers for resistance remains unclear. Though serious concerns of ML-resistance in D. immitis have existed for several years, no definitive studies that demonstrate this resistance under controlled conditions have yet been reported, and no assays for the detection of resistance have been validated.
The LMIA developed in this study was modified from those applied to gastrointestinal nematodes of ruminants for assessing drug-resistance status. Several other in vitro bioassays also exist for diagnosing ML resistance in gastrointestinal nematodes, the most commonly used being the larval development assay (LDA). However, the dissimilar biology of the filarioids prevents the application of this assay, and thus the LMIA was selected as the most viable and applicable technique. Extensive modification of the assay was required for optimal performance with filarial nematodes, which are, by comparison, highly sensitive to environmental exposure. In particular, four aspects of the D. immitis LMIA were the focus of optimization: (1) incubation time was adjusted to allow sufficient ML exposure while maintaining control parasite viability; (2) the medium and antibiotics were optimized for filarioid culture conditions; (3) the mesh size (25 lm) was selected for optimal discrimination of motile vs. non-motile D. immitis L 3 ; and (4) individual L 3 were counted and placed in wells of the assay plate rather than estimated numbers being aliquoted in known volumes of liquid, as is typically done in protocols for ruminant nematodes. This was done to reduce well-to-well variability given the relatively small numbers of D. immitis L 3 that could be readily obtained for each experiment.
The optimized assay reported here permitted a consistent highlevel migration in untreated control parasites and yielded consistent, reproducible dose-response data. This permitted us to calculate accurate IC 50 values for each drug examined. However, even at the highest ML concentrations that could be solubilized, complete inhibition of larval migration was rarely observed; the mean inhibition at 20 lM was 88.8% (SD = 6.89%) for eprinomectin and 70.0% (SD = 14.0%) for ivermectin. Therefore, it was not possible to accurately measure IC 95 values, leading to very wide 95% confidence intervals. Higher ML concentrations were not used owing to their poor solubility at the solvent level used (1% DMSO). Increasing the solvent level permits the use of higher ML concentrations, but this adversely affects parasite viability. Since it is critical that the untreated control parasites maintain high levels of viability throughout the entire experiment, this was not feasible. Any reduction in parasite viability in the no-drug control wells will increase variability and non-specific error, decreasing the consistency and accuracy of the data. The IC 50 usually is the most robust and accurate measure of the overall susceptibility of a population because data is collected at concentrations both above and below this value. This was also true of our data, thus the IC 50 was selected as the parameter of choice. Furthermore, with regard to the planned use of this assay, if an LOE case is not due to compliance failure, then all parasites in that dog would have established in the presence of regular ML prophylaxis. Consequently, all worms infecting the dog would be resistant, leading to reduced heterogeneity in the dose-response of the F2 generation. Thus, the IC 50 is the most logical and appropriate parameter for evaluating the susceptibility of any suspected-resistant D. immitis isolate with an in vitro assay. In contrast, there is usually high variability at the upper plateau of the dose-response, especially when 100% inhibition is not achievable. This tends to make the IC 95 a highly unstable parameter that is prone to variability-induced errors of interpretation.
Macrocyclic lactone resistance in H. contortus and Cooperia oncophora is well-established, and the LMIA has proven effective in discriminating resistant from susceptible isolates (Kotze et al., 2006;Demeler et al., 2010). Measuring these differences is also dependent upon how well dose-response data fit the statistical model being used, and in this study we found noticeably higher  R 2 values and smaller confidence intervals for IC 50 in assays with eprinomectin than with ivermectin. This disparity is not unusual, as in vitro responses in the LMIA have been reported to vary with the ML compound used (Kotze et al., 2006). This group also found that eprinomectin yielded superior results as compared to ivermectin in the LMIA. Gill and Lacey (1998) first reported that the optimal ML compound for use in an in vitro assay varies among assay type and parasite species, and thus needs to be determined empirically by experimentation for each assay-species combination. This finding demonstrates that the drug used in a particular in vitro bioassay has broad relevance and applicability for the entire anthelmintic class it represents. For example, in the LDA, the analog that provides the greatest discrimination between ML-susceptible and ML-resistant isolates of H. contortus and Trichostrongylus spp. is avermectin aglycone (Gill and Lacey, 1998;Kaplan et al., 2007), a drug that has poor efficacy in vivo and has not been developed as an anthelmintic product. Consequently, it is unimportant that eprinomectin is not used clinically for the prevention of heartworm infection. Future work will involve performing the LMIA with suspected-resistant D. immitis isolates, and the testing of additional ML compounds to determine whether eprinomectin or another analog is the best choice for discriminating heartworm populations of differing drug susceptibilities. The suitability of the LMIA for assessing ML susceptibility in filarioids must also be considered further. The LMIA is a motilitybased assay and the ML anthelmintics are known to cause paralysis in gastrointestinal nematodes of ruminants (Boisvenue et al., 1983;Folz et al., 1987). This rationale was the basis of the assay's development and validation as a diagnostic test for ML-resistance in ruminant nematodes. Given the extremely high ML sensitivity of D. immitis L 3 and L 4 stages, for which motility and migration are critical in both the initial infection of the canine host and subsequent tissue migration, we assumed that the LMIA would be the ideal assay for detecting ML resistance in this parasite. However, we observed that the ML concentrations required to induce an in vitro paralytic response were quite high. The preventive dose of ivermectin (6 lg/kg), which yields 100% clinical efficacy, produces peak plasma levels in the dog of approximately 3 ng/ml (Daurio et al., 1992). In contrast, the present study demonstrated an IC 50 value of 4.56 lM (3.99 lg/ml) for ivermectin, and even at 20 lM concentrations (17.5 lg/ml ivermectin; 18.3 lg/ml eprinomectin) we failed to effect total migration inhibition. In vitro concentrations more than 6000 times higher than those achieved in vivo could not come close to reaching the level of 100% inhibition. The disparity in drug level and the limited achievable efficacy challenge our assumption that the in vitro and in vivo responses of D. immitis to MLs are comparable and lead us to believe that the drug may be acting through at least two different mechanisms. Indeed, the host immune response may be integral to ML activity in D. immitis as indicated by research in other filarioids. In vitro treatment of Brugia malayi microfilariae with ivermectin was reported to interfere with protein secretion, which may unmask this parasite to the host immune system and allow its clearance (Moreno et al., 2010). Furthermore, studies with Acanthocheilonema viteae microfilariae reported that, while this filarioid is unaffected by nanomolar concentrations of ivermectin in vitro, it is subject to cell-mediated cytotoxicity when treated in the presence of host serum (Rao et al., 1987). Similar observations were reported for Litomosoides carinii, though for this parasite, treatment with ivermectin before exposure to immune cells appeared to elicit a cytotoxic response when worms were later cultured in the absence of drug (Zahner and Schmidtchen, 1994). These observations, both recent and old, suggest that the ML mechanism of action in filarioid parasites (clade III) may differ from that in the phylogenetically distinct gastrointestinal nematodes (clade V). If this is true, then it follows that in vitro bioassays developed for detecting ML resistance in gastrointestinal nematodes will not be effective in filarioids like D. immitis. Further research will investigate these issues, aiming to improve our understanding of the in vivo action of the MLs. We hope that further elucidation of the mechanism by which ML drugs kill these parasites will give us new insight into the effective measurement of ML susceptibility and, by extension, the diagnosis of resistance.


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Virus-Like Particles Containing the Tetrameric Ectodomain of Influenza Matrix Protein 2 and Flagellin Induce Heterosubtypic Protection in Mice

The ectodomain of matrix protein 2 (M2e) is highly conserved among influenza A viruses and can be a promising candidate antigen for a broadly cross-protective vaccine. In this study, a tetrameric M2e (tM2e) and a truncated form of flagellin (tFliC) were coincorporated into virus-like particles (VLPs) to enhance its immunogenicity. Our data showed that the majority of M2e in VLPs was presented as tetramers by introducing a foreign tetramerization motif GCN4. Intranasal immunization with tM2e VLPs significantly enhanced the levels of serum IgG and IgG subclasses compared to soluble M2e (sM2e) in mice. tM2e VLPs also induced higher M2e-specific T-cell and mucosal antibody responses, conferring complete protection against homologous influenza virus infection. The immunogenicity of tM2e VLPs was further enhanced by coincorporation of the membrane-anchored tFliC (tM2e chimeric VLPs) or coadministration with tFliC VLPs as a mixture, but not the soluble flagellin, inducing strong humoral and cellular immune responses conferring cross-protection against lethal challenge with heterotypic influenza viruses. These results support the development of tM2e chimeric VLPs as universal vaccines and warrant further investigation.

Introduction
Influenza A virus (IAV) is a negative sense single-stranded RNA virus responsible for annual seasonal epidemics worldwide and, occasionally, pandemics caused by emerging novel subtypes/strains derived by reassortment with avian or porcine viruses [1,2]. Current influenza vaccines are based primarily on antibody responses against the hemagglutinin (HA) or neuraminidase (NA) and provide strain-specific protection only [3,4]. Due to these limitations of current vaccines, it is crucial to establish a broadly cross-protective influenza vaccine, namely, universal vaccine. The appropriate presentation of an immunogen conserved in all influenza A viruses to the human immune system is important for an effective universal influenza vaccine.
The ectodomain of the influenza A M2 protein (M2e) is highly conserved among influenza A viruses and is considered to be a promising target for inducing cross-protection against different influenza A virus subtypes [5]. Some M2ebased vaccines protected mice from low-dose lethal virus challenge [6,7]. However, in most studies, M2e was not presented in its native tetrameric form or its membranebound environment. Since antibodies specific to conformational epitopes presented in quaternary structures may be more effective at binding M2 on cell surfaces [8], a tetrameric conformation-stabilized recombinant M2e presented in a membrane-anchored form, such as those incorporated into VLPs, was predicted to be more immunogenic than other M2e forms.
Toll-like-receptor-(TLR-) based immune adjuvants can induce efficient mucosal adjuvant activity [9]. The bacterial flagellin protein is the natural ligand of TLR-5 and is known as an effective adjuvant for enhancing immune responses [10,11]. Virus-like particles (VLPs) are known to be an effective vaccine platform which is egg independent and can elicit both humoral immune response and cellular immune response [11]. In our previous studies, we found that modified flagellin can be expressed effectively in a membrane-bound form and can be incorporated into M1-derived VLPs [12]. We also found that flagellin and four repeats of M2e can be fused together and incorporated into VLPs and induce strong humoral and cellular immune responses [10]. It is known that the central variable region of flagellin is essential for immunogenicity but not necessary for TLR-5 recognition, and the deletion of this region decreases the immunogenicity but retains its mucosal adjuvant function [13][14][15][16]. In this study, we designed a membrane-anchored tetrameric M2e protein stabilized by a foreign tetramerization sequence and incorporated the tM2e into influenza virus M1-based VLPs. Chimeric tM2e VLPs containing a truncated flagellin were also produced. We determined whether these VLPs induced broadly protective immunity in mice.

Ethics Statement.
The mice were housed and treated according to Emory University (Atlanta, GA) guidelines and all animal studies were approved by the Emory University Institutional Animal Care and Use Committee (IACUC).

Virus, Peptides, and Cells. Spodoptera frugiperda
Sf9 cells (Sf9, ATCC, CRL-1711) were maintained in suspension in serum-free SF900 II medium (GIBCO-BRL, Grand Island, NY) at 27 ∘ C in spinner flasks at a speed of 80 to 100 rpm. Madin-Darby canine kidney (MDCK) cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (HyClone, ThermoFisher, Rockford, IL). Mouse-adapted influenza A/Philippine/2/82 (H3N2) and A/PR/8/34 (H1N1) were prepared as lung homogenates from intranasally infected mice and were used for challenge studies. The M2e peptides were synthesized at GenScript (Piscataway, NJ, USA) as shown in Table 1. The purity of the peptide was above 95%. The peptide was dissolved in sterile water and stored at −20 ∘ C.

Construction of Membrane-Anchored tM2e and tFliC
Genes. A GCN4 sequence-stabilized tetrameric M2e (tM2e) construct was generated as described [17] but with a signal peptide (SP) encoding sequence from honeybee melittin and the transmembrane (TM) and cytoplasmic tail (CT) encoding sequences from the influenza A virus PR8 hemagglutinin (HA) in frame [12]. A membrane-anchored truncated flagellin (tFliC) encoding gene (deletion of variable region) was constructed as described previously [18] but with an influenza HA TM/CT. All constructs were confirmed by DNA sequencing analysis (Eurofins MWG Operon, Huntsville, AL). Recombinant baculoviruses (rBVs) expressing tM2e and tFliC were generated using the Bac-to-Bac protein expression kit (Invitrogen, Grand Island, NY) according to the manufacturer's instruction.

Cell Surface Expression of Membrane-Anchored tM2e.
The presence of membrane-anchored tM2e on cell surfaces was determined by a cell surface immune-assay. Two days postinfection with tM2e rBV and mock rBV (rBV expressing human immunodeficiency virus Gag) in 6-well plates, Sf9 cells were washed with phosphate-buffered saline (PBS) at 4 ∘ C and then incubated with 1 mL of NHS-SS-biotin dissolved in PBS (0.5 mg/mL) for 30 min at 4 ∘ C. Biotinylation was quenched by adding pre-cooled PBS containing 0.1 N glycine. After three washes with precooled PBS, the cells were then lysed with lysis buffer (150 mM NaCl/50 mM Tris HCl, pH 7.5/1 mM ethylenediamine tetraacetate/1% Triton X-100/1% sodium deoxycholate) and precipitated with avidin resins (Fisher scientific Inc., Rockford, IL) at 4 ∘ C overnight. After precipitation, resins were washed twice with lysis buffer plus 0.4% SDS and then mixed with 15 L sample buffer (125 mM Tris-HCl [pH 7.5] containing 4% SDS, 20% glycerol, plus 10% -mercaptoethanol) and heated at 95 ∘ C for 5 min before being analyzed by sodium dodecyl sulfatepolyacyrlamide gel electrophoresis (SDS-PAGE).

Cell Surface Immunogold Labeling and Electron
Microscopy of Infected Cells. At 60 hr postinfection, Sf9 cells were washed with 0.3 M HEPES containing 0.02 M lysine and were blocked in 0.2 M HEPES-1.5% bovine serum albumin (BSA) for 60 min at 4 ∘ C. After blocking, cells were incubated with mouse anti-M2e antibody (Abcam Inc., Cambridge, MA) at a 1 : 100 dilution at 4 ∘ C for 60 min. After washing, a 10 nm gold-conjugated goat anti-mouse antibody (Sigma-Aldrich, MO, USA) was added at a dilution of 1 : 100 for another 60 min. Cells were then washed and fixed with 1% glutaraldehyde in PBS for 30 min, incubated with 1% osmium tetroxide for 1 hour, and then stained with tannic acid. The samples were dehydrated and embedded in EMBED 812 (Electron Microscopy Sciences, Ft. Washington, PA). Samples were then stained with uranyl acetate and lead citrate and examined with a Philips (Mahwah, NJ) CM 10 electron microscope at the Robert P. Apkarian Integrated Electron Microscopy Core of Emory University.

Sample
Collections. Blood samples were collected at one week before immunization for preimmune sera and 2 weeks after each immunization for immune sera by retro-orbital plexus puncture. Sera were collected by a brief spin (5000 rpm for 5 min) after clotting (about 2 hours at room temperature). Mucosal samples were collected before challenge and 4 days after challenge. Nasal washes were collected by lavaging mouse nostrils repetitively with 1 mL of PBS containing 0.05% Tween 20 (PBST). For lung washes, individual mouse lungs were lavaged repetitively with 1 mL PBST. After a brief centrifugation (8000 rpm) for 10 min, supernatants were filtered through a 0.22 m filter and stored at −80 ∘ C for further assays. Lymphocytes from lung and spleen samples were collected from mice sacrificed 4 weeks after the final boost and were used for ELISPOT as described previously [12].
2.9. Antibody ELISA. M2e specific antibody (Ab) titers in immune sera were determined by ELISA as described previously [10,11] using synthesized M2e peptides (1 g/mL) as coating antigens. The highest dilution which gives an OD 450 of at least twice the standard deviation of that of the naïve group at the same dilution was designated as the Ab endpoint titer. To evaluate the cross-reactivity to variant M2e-sequence peptides, immune sera were tested for binding to M2e peptides derived from Phi/82, PR/8, CA/09, and VIET/04, respectively, by ELISA. The optical density at 450 nm was used to compare the level of reactivity. Antibodies recognizing native M2 protein were determined by using cell surface ELISA. M2-expressing MDCK cells were maintained in DMEM media with 7.5 g/mL of puromycin (Invitrogen, Carlsbad, CA), 5 M of amantadine (Sigma, St. Louis, MO) and 10% FBS (Invitrogen, Carlsbad, CA) at 37 ∘ C in 5% CO 2 . Confluent M2-expressing MDCK monolayer cells were fixed by 0.05% glutaraldehyde and 10% buffered formalin (Sigma, St. Louis, MO) for 30 min at room temperature and were used to determine Ab levels binding to M2 expressed on cell surfaces by ELISA as described [12].

Cytokine ELISpot.
Interferon gamma (INF-) and interleukin 4 (IL-4) secreting T cells from immunized mouse splenocytes or lung cells were evaluated using ELISpot kits (eBioscience, San Diego, CA) according to the manufacturer's instructions [12]. Anti-mouse IFN-or IL-4 Abs was used to coat Multiscreen 96-well filtration plates (Millipore, Billerica, MA). A volume of 100 L freshly isolated splenocytes (1 × 10 7 cells/mL) were added to each well and stimulated with M2e peptide (10 g/mL). The plates were incubated for 40 h at 37 ∘ C with 5% CO 2 . The development and counting of ELISpot were performed following the manufacturer's procedures.

Lung Viral Titers.
Whole lungs were collected at day 4 postinfection (p.i.) and ground using cell strainers (BD Falcon, Franklin Lakes, NJ). Lung homogenates were centrifuged at 1000 RPM for 10 min to remove tissue debris. MDCK cell-based plaque assay was used for lung virus titration as described previously.

Statistical Analysis.
Comparisons among vaccinated groups were performed using a two-tailed Student's t-test. The analyses were done by using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). P values of less than 0.05 ( < 0.05) were considered to be statistically significant.

Preparation and Characterization of VLPs.
To improve the immunogenicity of M2e, a modified tetrameric M2e (tM2e) construct was designed as showed in Figure 1(a). A modified flagellin with a central variable region truncation and a membrane anchor from influenza HA was designed as a molecular adjuvant, as described before with slight modifications [18]. To determine the role of tM2e in inducing cross protection against heterologous virus, we produced influenza VLPs containing the tM2e protein. To confirm the tetrameric structure of tM2e after expression and purification, we used the chemical cross-linker Bis[sulfosuccinimidyl] suberate (BS3) to fix its polymeric state. Following cross-linking, SDS-PAGE analysis revealed a major band with a molecular mass of 28 kDa representing the M2e tetramer and a band with a molecular mass of 14 kDa representing the dimers (Figure 1(b), lane 2). We observed only one band with a molecular mass of 7 kDa representing the M2e monomer without the addition of BS3 (Figure 1 of insect cells, demonstrating that the GCN4-stabilized tM2e was expressed in a tetrameric form and could be transported onto the cell surface [17]. The lower band in Figure 1 content in VLPs was evaluated using Western blotting as described [19]. Recombinant M2e protein (Lanes 1, 2, and 3 and 4, 15, 30, 60, and 120 ng, resp.) was used as standard, and tM2e VLPs (Lane 6: 5 g total protein) and tM2e/tFliC VLPs (Lane 5: 5 g total protein) were loaded and detected. Amount of M2e protein incorporated in M2e VLP was calculated by spot densitometry analysis using serial diluted rM2e protein as a standard as shown in Figure 1 as described [19]. We also use soluble flagellin as standard and check the tFlic concentration in tFlic VLPs and used same dose as soluble flagellin. Furthermore, the presence of tM2e in the VLPs and tM2e VLP budding from sf9 cell membrane were confirmed by examining sections of cell surface after immunogold labeling as shown in Figure 2. Both tM2e VLPs and tM2e/tFliC VLPs showed spherical shapes with gold patches on their surfaces, demonstrating the incorporation of tM2e into VLPs and tM2e VLP budding from insect cells.

Tetrameric M2e VLPs Induced Strong Humoral Responses.
To determine if i.n. immunization with tM2e VLPs could induce enhanced humoral responses, immune sera were evaluated for antigen-specific IgG titers using ELISA with M2e peptides as coating antigens, or cell surface ELISA using M2-expressing MDCK cells. As shown in Figure 3(a), M2especific IgG endpoint titers in sera of G2 (tM2e VLPs), G3 (tM2e/tFliC VLPs), G4 (tM2e VLPs + tFliC VLPs), and G5 (tM2e VLPs + soluble flagellin) were approximately 10-to 30-fold higher than that of the mouse group immunized with only soluble tM2e ( < 0.05) (Figure 3(a)). The levels of M2e-specific IgG1 and IgG2a were also significantly higher than those of mice receiving only tM2e (Figures 3(b)  and 3(c)). Flagellin showed a significant adjuvant effect on the magnitude of M2e-specific Ab responses when either integrated into tM2e VLPs or when tFliC VLPs were mixed with tM2e VLPs. As shown in Figure 3, the levels of IgG, IgG1, or IgG2a in sera of G3 (tM2e/tFliC VLP) and G4 (mixture of tM2e VLPs and tFliC VLPs) were significantly higher than G2 which was immunized with tM2e VLPs alone. To provide more details of the comparison of the antibody titer between each group, endpoint titers of each group were shown in Supplemental Material (Tables 1-4 of IgG2a (IgG1/IgG2a ratio, 0.89 and 0.7, resp.) compared to tM2e VLPs (G2) (IgG1/IgG2a ratio, 1.23). Immune sera also showed similar binding reactivity to native M2 expressed on MDCK cell surfaces, suggesting that antibodies induced by tM2e VLPs can recognize conformational epitopes on native M2. These data show that tM2e VLPs can elicit strong humoral responses and that membrane-anchored truncated flagellin shows a strong adjuvant effect.
Although M2e is highly conserved in influenza A viruses, some amino acid substitutions occur in different strains and subtypes. To evaluate the cross-reactivity of M2e-specific Abs induced by tM2e VLPs, sera from mice immunized i.n. with tM2e VLPs were assayed using ELISA with variant M2e peptides as coating antigens. As shown in Table 1, the M2e sequence in tM2e VLPs is the same in human viral M2e consensus and in Phi/82 H3N2 M2e, whereas there are 1, 4, and 3 amino acid differences, respectively, in M2e of PR/8, CA/09 and VIET/04 viruses. As demonstrated in Figure 4, M2e Abs bound strongly to the M2e peptides of Phi/82 and PR/8. The levels of Ab binding were significantly lower in VIET/04 and CA/09 M2e peptides ( < 0.05). These results demonstrate that the assembly of the tM2e antigen into VLPs can significantly enhance the immunogenicity of tM2e and can induce M2e-specific antibodies with some crossreactivity to variant M2e sequences. These results also show that when M2e and flagellin are combined together in VLPs (G3) or tM2e VLPs and flagellin VLPs are mixed together (G4), systemic responses were enhanced more than tM2e VLPs mixed with soluble flagellin protein after immunization (G5), which indicate that the membrane-anchored truncated flagellin shows a stronger adjuvant effect than the soluble form in induction of serum IgG responses.

Tetrameric M2e VLPs Induce Robust Mucosal Antibody
Responses. A strong mucosal antibody response is associated with the prevention of viral entry in the upper respiratory tract of mice [20]. To evaluate whether i.n. immunization  . Each group has 6 mice. The asterisk ( * ) indicates a significant difference between G2 and G1, G3 and G1, G4 and G1, and G5 and G1. The "+" indicates a significant difference between G3 and G2, G4 and G2, and G5 and G2.
with tM2e VLPs could induce mucosal Ab responses, we measured M2e-specific IgA and IgG Abs from lung and nasal washes by ELISA in mice prechallenge and 4 days after challenge ( Figure 5). As shown in Figures 5(a) and 5(  IgA levels increased, as well as nasal wash IgA, and there was no significant IgG change between prechallenge and postchallenge nasal washes ( Figure 5). Similarly, significantly higher levels of IgA were observed in nasal washes of prechallenge or postchallenge mice immunized with tM2e VLP, tM2e/tFliC VLP, tM2e VLP + tFliC VLP, or tM2e VLP + soluble flagellin ( < 0.05). These data demonstrate that tM2e VLPs stimulate M2e-specific mucosal Ab responses in mice, and incorporating the membrane-anchored flagellin in VLPs as adjutants can further stimulate mucosal antibody secretion.

Tetrameric M2e VLPs Activate M2e-Specific T-Cell
Responses. T-cell responses are important for the generation and regulation of an effective immune response and are known to contribute to broad cross-protective immunity [21,22]. IFN-and IL-4 secreting cells in both the spleens and lungs of immunized mice were evaluated by cytokine ELISpot as described previously [11]. As shown in Figure 6, mice of groups G2, G3, G4, or G5 showed significantly higher IFNsecreting cell populations in the spleens and lungs after stimulation with M2e peptide or tM2e VLPs compared with mice immunized with soluble tM2e (Figures 6(a) and 6(b), < 0.05). A higher frequency of IL-4 secreting cells was also detected in the spleens of immunized mice in G2, G3, G4, or G5 compared to that of mice vaccinated with soluble tM2e (Figure 6(c), < 0.05). However, only G2, G3, and G4 had higher frequencies of IL-4 secreting cells in the lungs after stimulation with tM2e VLPs and only background levels of cytokine secreting cells were detected after M2e peptide stimulation (Figure 6(d)). These results provide evidence that i.n. immunization with tM2e VLPs induces enhanced M2e specific T-cell immune responses.

Tetrameric M2e VLPs Protect Mice from Lethal Virus
Challenge. To evaluate the protective efficacy of tM2e VLPs, immunized mice were infected with 5LD 50 of Phi/82 H3N2 virus or PR/8 H1N1 virus 4 weeks after the final boost immunization. Mouse body weight loss and survival were monitored for 14 days. Day 4 postinfection was chosen for evaluation of lung virus titers because naive mice were previously shown to have substantial titers of virus in the lungs at that time point [23].
As shown in Figure 7, all mice immunized with tM2e protein alone, soluble flagellin protein alone or naive groups died by 7 to 9 days p.i. (Figures 7(b), and 7(d)) with high titers of viruses in the lungs (Figures 7(a) and 7(c)). G1 (tM2e protein), G2 (tM2e VLP), G5 (tM2e VLP + soluble flagellin), and G6 (soluble flagellin protein) did not show substantial virus reduction in the lungs compared with the control ( > 0.05). In contrast, mice immunized with tM2e/tFliC VLPs or tM2e VLP + tFliC VLPs had significant lower virus titers in their lungs after Phi/82 or PR/8 challenge ( < 0.05) and exhibited reduced morbidity compared with mice in the tM2e protein group. All mice in G3 and G4 survived the lethal challenge with homologous Phi/82 H3N2 virus or heterosubtypic PR/8 H1N1 virus. These results indicate that i.n. immunization of mice with tM2e VLPs with flagellin as an adjuvant significantly reduces virus titers in the lungs and completely protects mice against severe disease and death from lethal infection with the homologous virus or heterosubtypic virus.

Discussion
M2e is considered to be a promising target for inducing crossprotection because it is highly conserved among influenza A viruses [5]. However, the immunogenicity of M2e is very low due in part to its low incorporation level into influenza virus particles and its small ectodomain, which may be sterically blocked by HA and NA on the surface of the virus. These properties of native M2e limit its ability to be sensed by host immune cells. In this study, we found that tM2e was incorporated into virus-like particles at high levels without the presence of HA and NA and thus could be delivered to and effectively sensed by immune cells. Since antibodies specific to conformational epitopes presented in quaternary structures may be more effective at binding native M2 [8], a tetrameric conformation-stabilized recombinant M2e was predicted to be more immunogenic than other M2e forms. The tetrameric M2e that we produced was stabilized by the addition of tGCN4 [17] and a C-terminal membrane anchor from the A/PR8 influenza virus HA [10] and was expressed efficiently in a recombinant baculovirus protein expression system in insect cells. Our results demonstrated that intranasal vaccination with tM2e VLPs induces high levels of humoral and mucosal M2e-specific antibody and Tcell responses compared to those observed with the soluble tM2e. Furthermore, these Abs were shown to recognize native M2 on cell surfaces.
VLPs is an effectve platform to increase the immunogenicity of antigen [10]. Besides, we have observed that the TLR5 ligand flagellin can be incorporated into influenza VLPs and is an effective adjuvant for preventing mucosal infection of influenza viruses [11,12]. However, there are questions about whether the immunogenicity of flagellin is a disadvantage for its use as an adjuvant [12,24]. Considering these, we modified flagellin by truncating the variable region which is essential for immunogenicity but not necessary for its TLR5 binding activity and incorporated it into influenza tM2e VLPs by fusing a membrane-anchoring sequence from HA. We found that the membrane-anchored truncated flagellin shows a stronger adjuvant effect than the soluble form in induction of humoral immune responses when animals were immunized intranasally. The same amount of soluble flagellin also showed reduced adjuvant effect when compared with tFliC VLPs, which suggest that different mechanisms may be involved in these two different forms.
Flagellin can promote murine antibody responses by either membrane receptor TLR5-mediated activation of NF-B or cytosolic receptor NLRC4-mediated activation of the inflammasome [25]. VLPs are similar to native virus in size and can enter cells to release contents of the core-like particle into the cytoplasm of the cell [11]. Flagellin-containing M2e VLPs can enter cells, react with the flagellin cytosolic receptor NLRC4 (also referred to as IPAF), and drive rapid generation  Figure 6: Cellular immune responses. Cellular immune responses were assessed with cells from spleens and lungs of immunized mice. Cells from spleens ((a), (c)) and lungs ((b), (d)) were stimulated with M2e peptides and M2e VLPs for 40 hours, and cytokine-secreting cell colonies were determined by ELISPOT assay. Each group has 3 mice. The asterisk ( * ) indicates a significant difference between G2 and G1, G3 and G1, G4 and G1, and G5 and G1. The "+" indicates a significant difference between G3 and G2, G4 and G2, and G5 and G2.
of inflammasome cytokines such as IL-1 and IL-18 to provide a sufficient second signal. The cytosolic innate signaling drives the T-cell proliferation necessary to generate antibodies to both flagellin itself and a coadministered antigen [25]. Soluble flagellin can recognize flagellin receptor TLR5 on the cell surface and activate NF-b [25]. In our experimental groups, soluble flagellin administrated with M2e VLP could protect mice from homologous virus infection but not heterosubtypic virus infection, whereas flagellin-containing M2e VLPs could protect mice from both homologous and heterosubtypic virus infections. This phenomenon suggests that NLRC4 dependent pathway may play a dominant role in the promotion of antibody responses by flagellin-containing VLPs. Furthermore, we found that tFliC-containing VLPs preferentially activated Th1-responses with a lower IgG1/IgG2a ratio, which is similar to the membrane-anchored full-length flagellin and flagellin in its native surface-bound context on live Salmonella [12,26]. However, the mechanism of tFliC on Ig isotype switching is still unknown.
Our aim was to combine the advantage of VLPs as a vaccine platform and flagellin as an adjuvant to increase the immunogenicity of the conserved M2e and induce crossprotective immunity to homo-/heterologous influenza viral infections. From our challenge data we can see that even  with high level of Ab titers, M2e VLPs induce limited protection from heterosubtypic virus infection. One possible reason is that so far only a single human CTL epitope has been identified for M2e inducing memory CTL activity [27], and M2e specific antibody does not have neutralization effect but they have weak protection via antibody-dependent NK cell activity [28]. In fact, in our experiment, tFliccontaining VLPs could enhance immune response via the NLRC4 pathway since VLPs can enter into the cells. So the antibody-dependent cell-mediated cellular cytotoxicity might be involved in the protective effect of heterosubtypic virus infection. The mucosal immune system is the first immunological barrier against viruses that invade the body via the mucosal surface [29]. M2e-based vaccines were previously reported to induce mucosal immune response when delivered by the i.n. route [30][31][32]. Several groups demonstrated that IgA in upper respiratory tract secretions plays a major role in antiviral immunity [33][34][35]. The local production of IgG is also an important component of the immune responses following mucosal immunization or infection [36,37]. We found that high levels of sIgA and IgG were induced in both nasal mucosal and lung surfaces after i.n. administration of tM2e VLPs. The consistency of these antibody responses to the observed protection indicates that mucosal antibodies may be one of the immune correlates of protection. A combination of enhanced mucosal immunity and antibody-dependent cellmediated cellular cytotoxicity may be protecting mice from lethal challenge from homo/hetero strains.
Although M2e is relatively conserved in all influenza A viruses, variation of M2e sequences in different viruses may indicate the different protection efficacy of M2e vaccines to various virus infection. In our study, tM2e VLPs showed complete protection of mice from Phi/82 (H3N2) virus infection; it still cannot protect mice from PR/8 (H1N1) virus infection which may be associated with the 1-aa substitutions in the M2e of PR/8. Indeed, our ELISA binding assay illustrated that even a 1-aa variation could impact the binding avidity of M2e-specific Abs with synthesized peptides. However, it is noteworthy that tFlic-containing VLPs showed complete protection of mice from either Phi/82 (H3N2) virus infection or PR/8 (H1N1) virus infection. Therefore, it is worth to further investigate those strategies that could enhance the cross-protection of M2e vaccines, for instance, incorporating representative M2e sequences from different subtypes into one construct, or coexpressing with HA or NA antigens, as well as combining with better adjuvant strategy.

Conclusions
M2e is one of the target antigens for a universal vaccine against influenza. Strategies like administration in naturally conformational form carried by VLPs and adjuvanted by bioactive factors demonstrate significant improvement of both immunogenicity and protective efficacy of M2e. Our results support the promising application of tM2e cVLPbased universal vaccines and warrant further investigation.


s2fieldsofstudy: Biology Psychology

text:
Putative physiological mechanisms underlying tDCS analgesic effects

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique that induces changes in excitability, and activation of brain neurons and neuronal circuits. It has been observed that beyond regional effects under the electrodes, tDCS also alters activity of remote interconnected cortical and subcortical areas. This makes the tDCS stimulation technique potentially promising for modulation of pain syndromes. Indeed, utilizing specific montages, tDCS resulted in analgesic effects in experimental settings, as well as in post-operative acute pain and chronic pain syndromes. The promising evidence of tDCS-induced analgesic effects raises the challenging and complex question of potential physiologic mechanisms that underlie/mediate the accomplished pain relief. Here we present hypotheses on how the specific montages and targets for stimulation may affect the pain processing network.

Transcranial direct current stimulation (tDCS) is a noninvasive neuromodulation technique (Nitsche and Paulus, 2000) that delivers electrical current of relatively low intensity (1 or 2 miliamperes over an area of about 20 to 35 cm 2 ) painlessly through the skull to selected areas of the brain, and induce changes in excitability and activation of brain neurons and neuronal circuits. An important, and perhaps primary, mechanism of tDCS is a subthreshold modulation of neuronal resting membrane potential. Stimulation of severalminute duration results in a polarity-dependent induction of glutamatergic calcium-dependent neuroplasticity, which shares some aspects with long-term potentiation, and depression . The effects of tDCS on cortical excitability are polarity-dependent. Anodal tDCS enhances, while cathodal tDCS diminishes excitability, within certain parameters of stimulation duration and strength Paulus, 2000, 2001;. Too long, or strong stimulation, however, may have an opposite effect, resulting in diminished excitability after the anodal stimulation and enhanced excitability after the cathodal tDCS (Batsikadze et al., 2013;Monte-Silva et al., 2013). In addition, recent evidence suggests that tDCS interacts with various cerebral neurotransmitter systems, and is mediated by dopamine, acetylcholine, serotonin or GABA (Nitsche et al., 2004a(Nitsche et al., ,b,c, 2006(Nitsche et al., , 2009Kuo et al., 2007;Terney et al., 2008). Moreover, tDCS has been shown to facilitate changes in brain-derived neurotrophic factor (BDNF; Fritsch et al., 2010) that is a distinct marker of neuronal plasticity and notably has been associated with pain processing (Stefani et al., 2012).
The effects and outcome of tDCS depend on the area of the brain that is stimulated (e.g., Nitsche et al., 2007). Beyond regional effects under the electrodes, activity alterations of interconnected remote cortical and subcortical areas have also been described (Polania et al., 2011;DaSilva et al., 2012). This makes the tDCS stimulation technique potentially promising for modulation of pain syndromes, which include pathological alterations of activity, and excitability of a multitude of interconnected areas. Different interwoven cortico-subcortical pain-related networks, so-called Pain Matrix, cover sensory-discriminative, affective, and vegetative aspects of pain processing. The main components of the sensory-discriminative pain processing network are the spinothalamic tract, the lateral thalamus, somatosensory areas, and the posterior insula (Moisset and Bouhassira, 2007). The affective component of pain has been related to anterior insular, and cingulate cortices, as well as prefrontal areas. Vegetative, and neuroendocrine effects of pain perception are closely linked to various subcortical regions, such as amygdala, hypothalamus ventral tegmental area and others (Hsieh et al., 1995;Zaghi et al., 2009). Neuroplastic alterations of connectivity between these areas might contribute to chronification of pain.
Several specific tDCS montages have been probed, which resulted in analgesic effects: (a) excitability-enhancing (anodal) tDCS delivered over the primary motor cortex (e.g., Fregni et al., 2006a,b;Fenton et al., 2008;Kuhnl et al., 2008;Knotkova et al., 2013), typically with the anode positioned over M1 contralateral to the affected side and cathode over the ipsilateral supraorbital region in case of unilateral pain; or the anode over M1 of the Frontiers in Human Neuroscience www.frontiersin.org September 2013 | Volume 7 | Article 628 | 1 dominant hemisphere and the cathode over the supraorbital region contralateral to the anode in case of bilateral pain; (b) excitability-diminishing (cathodal) tDCS over the somatosensory cortex Knotkova et al., 2009) [the cathode over S1, the anode over the contralateral supraorbital region, with the same consideration of pain localization as described above]; (c) anodal tDCS over the left dorsolateral prefrontal cortex (DLPFC; Riberto et al., 2011;Valle et al., 2009) [the anode over DLPFC corresponding with the F3 electrode position of the 10-20 international EEG system, the cathode over the contralateral supraorbital region]; (d) combined anodal left DLPFC and cathodal tDCS of contralateral somatosensory cortex [the cathode over the gut representation area of the right S1] (Borckardt et al., 2011).
In the available studies, the assessment of analgesic effects elicited with these montages in subjects with bilateral pain has not systematically compared the pain intensity separately at each side, and thus it is unclear if the effect of the stimulation was unilateral or bilateral. However, as noted by Antal et al. (2010), there is evidence that tDCS of M1 induces widespread changes in cortical activity and can induce changes in activity of the contralateral hemisphere.
Analgesic effects have been explored in experimental settings (experimentally induced pain in healthy subjects), as well as in post-operative acute pain and chronic pain syndromes in clinical settings. The analgesic effects have been shown to be cumulative, and therefore a majority of clinical trials of tDCS encompassed the stimulation on several (usually 5, rarely 10) consecutive days. For example, anodal M1 tDCS over five consecutive days resulted in a significant decrease of pain intensity after spinal cord injury (Fregni et al., 2006a), and similar results were observed in chronic neuropathic pain due to multiple sclerosis (Mori et al., 2010), chronic pelvic pain (Fenton et al., 2009) and pain of various origin (Antal et al., 2010;Knotkova et al., 2013). Stimulation over the prefrontal cortex resulted in significant pain relief after 10 but not 5 sessions (Valle et al., 2009). Meta-analysis of analgesic effects in the existing studies is thoroughly discussed in a Cochrane systematic review by O'Connell et al. (2011). Overall, a significant heterogeneity among studies was noted, and a sub-group evaluation of tDCS applied to the motor cortex suggested superiority of active stimulation over sham (SMD −0.59, 95% CI −1.10 to −0.08).
The evidence of tDCS-induced analgesic effects raises the challenging and complex question of potential physiologic mechanisms that underlie/mediate the accomplished pain relief. Here we develop hypotheses on how the specific montages and targets for stimulation may affect the pain processing network.

MODULATION OF THE SENSORY-DISCRIMINATIVE PAIN PROCESSING CHANGES IN THALAMIC ACTIVITY
Thalamic activity is crucial for processing and filtering of nociceptive signals on the pathways ascending to the cortical part of the pain matrix, and the thalamus also receives direct input from descending cortico-thalamic pathways originating in the primary motor cortex. Notably, anodal tDCS over M1 has been shown to increase functional coupling between ipsilateral M1 and thalamus (Polania et al., 2011) and therefore it is likely that the analgesic effects observed after the facilitatory motor cortex stimulation are at least partially attributable to modulation of thalamic activity. Indeed, changes in regional cerebral blood flow following epidural motor cortex stimulation (Peyron et al., 1995;Garcia-Larrea et al., 1999;Garcia-Larrea and Peyron, 2007) indicated that stimulation of the motor cortex may trigger rapid and phasic activation in the lateral thalamus (which receives direct input from the motor area), followed by parallel or secondary activation of medial thalamic regions, and the anterior cingulate gyrus, the insula and the upper brain stem. (Garcia-Larrea et al., 1999). Interestingly, the blood flow change in the lateral thalamus has not significantly correlated with patient's perceived pain relief and although important as a trigger of further events, the activation of the lateral thalamus is not a sufficient condition for clinical pain-relieving effects (Garcia-Larrea et al., 1999). However, neuronal inactivation in response to motor cortex stimulation was detected in thalamic sensory neurons, specifically in ventral posterolateral nuclei and centromedian-parafascicular thalamic complex, and the inactivating effect was particularly observed for neurons responsive to nociceptive peripheral stimulation (Pagano et al., 2012). It can be speculated that the inhibition of the sensory thalamic nuclei and the activation of the lateral (motoric) thalamic area after the motor cortex stimulation may be functionally related, the lateral thalamus receiving the input from the motor cortex and inhibiting the thalamic sensory neurons that are involved in the transmission of nociceptive signals from the periphery.

MOTOR-CORTEX-DRIVEN INHIBITION OF THE SOMATOSENSORY CORTEX
As there is a direct connection between the primary motor cortex and primary somatosensory cortex via cortico-cortical pathways in the human brain, it is possible that stimulation of the motor cortex directly inhibits the activity in the somatosensory cortex. By these means, recent work by Chiou et al. (2012) on animal models demonstrated that motor cortex stimulation blocked the transmission of somatosensory information to the primary somatosensory cortex. In the experiment, epidural motor cortex stimulation, but not stimulation outside of the motor cortex, lead to suppression of the ipsilateral somatosensory evoked potentials. However, these findings have to be interpreted with caution as the stimulation was delivered at suprathreshold level, and therefore the effects cannot be directly extrapolated to the subthreshold tDCS stimulation. Interestingly, a study of the somatosensory cortex in rats (Choi and Callaway, 2011) has shown the existence of inhibitory neurons in the somatosensory cortex that receive direct monosynaptic input not only from distant areas such as thalamus, but also from the ipsilateral motor cortex.

DIRECT INHIBITION OF THE SOMATOSENSORY CORTEX
Cathodal tDCS is thought to have a direct excitability-reducing effect on the S1 area. Since hyperexcitability within S1 in chronic pain syndromes, such as facial neuropathic pain or carpal tunnel syndrome has been clearly documented in recent neuroimaging studies, tDCS-generated reduction of this pathological excitability alteration should be beneficial. Moreover, thickening of neuronal Frontiers in Human Neuroscience www.frontiersin.org September 2013 | Volume 7 | Article 628 | 2 layers in the somatosensory cortex has been observed in chronic migraineurs (DaSilva et al., 2007), which might be a hint for structural neuroplastic alterations of the respective area due to its pain-related hyperactivity of this area. It has been suggested that repeated/long-term down-regulation of nociceptive activity in S1, which could be also induced by tDCS, may result in normalization of this maladaptive change.

ACTIVATION OF THE PREFRONTAL CORTEX
Stimulation of the prefrontal cortex has been associated with a modulation of a large neuronal network related to the limbic system, including the cingulate gyrus and parahippocampal areas (Mottaghy et al., 2000;Catafu et al., 2001). The dopaminergic and serotoninergic circuits of the frontal and prefrontal cortex and related subcortical areas mediate attentional control, impulsivity, working memory, decision-making, as well as mood regulation and emotional processing. Notably, activation of the brain structures associated with emotional appraisal of pain in condition of the epidural motor cortex stimulation correlated with subjectively reported pain relief (Peyron et al., 1995;Garcia-Larrea et al., 1999) and it is thought that neuromodulation modifying emotional appraisal of pain and pain experience is directly related to clinical analgesic effects of the neuromodulatory interventions (Garcia-Larrea et al., 1999). Indeed, tDCS stimulation of the prefrontal dorsolateral cortex increased pain thresholds in healthy subjects  and relieved chronic pain (Valle et al., 2009).

OPIOID RELEASE
Interestingly, a recent work by DosSantos et al. (2012) has shown that a single session of anodal tDCS over the motor cortex results in reduction of mu opioid receptor binding of an exogenous receptor ligand in the pain matrix, suggesting that the analgesic effect of M1-tDCS may possibly be due to a direct increase of endogenous opioid release . The authors suggest that the decreased binding of the exogenous ligand was possibly due to receptor occupancy by enhanced release of endogenous opioids. The reduction was detected in numerous cortical and subcortical structures of the pain matrix, such as nucleus accumbens, anterior cingulate cortex, insula and thalamus, and was accompanied by an increased threshold for experimentally induced cold pain. Although opioid analgesic effects are known to relate to both the emotional-as well as sensory dimension of pain, no significant changes in clinical pain levels were elicited after a single tDCS session, suggesting that the immediate opiodergic effects of a single tDCS application are subclinical, and repeated application might be necessary to get clinically meaningful results.

CONCLUSIONS AND IMPLICATIONS FOR FUTURE RESEARCH
The findings suggest that multiple physiologic mechanisms mediate the analgesic effects of tDCS, involving changes in both the perceptual processing of pain and the emotional component of pain experience. However, the mechanisms and their translation into predicable clinical outcomes are far from being fully understood. Future studies are needed to expand understanding of tDCS-induced analgesic mechanisms and to address the presented hypotheses of tDCS effects on the pain-processing network. Extrapolating from studies of the epidural motor cortex stimulation, changes of the thalamic activation after tDCS may be determined via the regional cerebral blood flow evaluation in the thalamus and related regions after a single-and multiple tDCS stimulation of the motor cortex, including explorations of the association between the thalamic activation changes and pain relief. Future studies addressing the hypothesis of the tDCS-generated analgesic effects due to motor-cortex-driven inhibition of the somatosensory cortex may utilize evaluations of the somatosensory potentials, exploring suppression of the somatosensory evoked potentials after the anodal tDCS stimulation of the ipsilateral motor cortex. Moreover, studies of tDCS combined with functional imaging (fMRI) with regard to the inhibitory (cathodal) stimulation of the somatosensory cortex as well as the anodal tDCS in both the experimentally induced-and spontaneous chronic pain may provide further insight into the tDCS effects on the pain matrix. Beyond the exploration of regional effects, functional imaging data might also be helpful to explore stimulation-dependent alterations of the pain-related cerebral network, via functional connectivity analysis. The latter approach will be also relevant to explore specific effects of different stimulation paradigms on the above-mentioned discernable components of the pain matrix. Further, future studies are needed to systematically elucidate the impact of the stimulation parameters on the analgesic outcomes, including aspects related to stimulation intensity, strength, repetition rate and timing, as well as electrode positions and stimulation polarity, because a critical aspect of the future impact of tDCS in pain management is the optimization of the stimulation protocols with regard to specific patient populations.