Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

Homozygous staggerer mice (sg/sg) display decreased and dysfunctional retinoic acid receptor-related orphan receptor ␣ (ROR␣) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in sg/sg mice. Moreover, the sg/sg mice were characterized by reduced adiposity (associated with decreased fat pad mass and adipocyte size). Candidate-based expression profiling demonstrated that the dyslipidemia in sg/sg mice is associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This is consistent with the reduced serum lipids. The molecular mechanism did not involve aberrant expression of LXR and/or ChREBP. However, ChIP and transfection analyses revealed that ROR␣ is recruited to and regulates the activity of the SREBP-1c promoter. Furthermore, the lean phenotype in sg/sg mice is also characterized by significantly increased expression of PGC-1␣, PGC-1␤, and lipin1 mRNA in liver and white and brown adipose tissue from sg/sg mice. In addition, we observed a significant 4-fold increase in ␤ 2 -adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional ROR␣ expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type but not sg/sg mice exhibited a ϳ20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues are involved in decreased adiposity and resistance to dietinduced obesity in the sg/sg mice, despite hyperphagia. In conclusion, we suggest this orphan nuclear receptor is a key modulator of fat accumulation and that selective ROR modulators may have utility in the treatment of obesity.The spontaneously arising mouse mutant, staggerer (sg), 4 was initially described and analyzed several decades ago (1).The homozygous mice display ataxia, cerebellar defects, tremor, and imbalance. Subsequently, the gene encoding retinoic acid receptor-related orphan receptor ␣ (ROR␣) (2, 3) was mapped to mouse chromosome 9 in the immediacy of the sg locus (4, 5). ROR␣ is an orphan member of the nuclear receptor superfamily (6). Several studies have identified cholesterol sulfate (and derivatives) as potential ligands for this nuclear receptor (7, 8), however, further studies are required to resolve this issue.The sg mutation has been shown to be an intragenic deletion in the coding region of ROR␣, removing an exon downstream of the DNA binding domain (4,5). This results in a frameshift mutation that affects the co-expressed isoforms ROR␣1 and -␣4 (5). ROR␣ transcript levels are significantly reduced in staggerer (sg/sg) mice, and it has been clearly demonstrated that the sg phenotype is associated with the expression of dysfunctional ROR␣ (9). Moreover, ROR␣-deficient mice display many aspects of the staggerer phenotype (9, 10). Mamontova et al. (11) demonstrated that male and female homozygous staggerer (sg/sg) are charac...

A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions (HCVcc) with various activities. Although nonneutralizing HC33 HMAbs were isolated, they had lower binding affinities than neutralizing HC33 HMAbs. These antibodies could be converted to neutralizing antibodies by affinity maturation. Unidirectional competition for binding to E2 was observed between HC33 HMAbs and HMAbs to aa 434 to 446. When HMAbs to aa 434 to 446, which mediated neutralization, were combined with neutralizing HC33 HMAbs, biphasic patterns in neutralization were observed. A modest degree of antagonism was observed at lower concentrations, and a modest degree of synergism was observed at higher concentrations. However, the overall effect was additive neutralization. A similar pattern was observed when these antibodies were combined to block E2 binding to the HCV coreceptor, CD81. These findings demonstrate that both of these E2 regions participate in epitopes mediating virus neutralization and that the antibodies to aa 412 to 423 and aa 434 to 446 do not hinder their respective virus-neutralizing activities.

The staggerer mice carry a deletion in the ROR␣ gene and have a prolonged humoral response, overproduce inflammatory cytokines, and are immunodeficient. Furthermore, the staggerer mice display lowered plasma apoA-I/-II, decreased plasma high density lipoprotein cholesterol and triglycerides, and develop hypo-␣-lipoproteinemia and atherosclerosis. However, relatively little is known about ROR␣ in the context of target tissues, target genes, and lipid homeostasis. For example, ROR␣ is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for ϳ40% of total body weight and 50% of energy expenditure. This lean tissue is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. In particular, the role of ROR␣ in skeletal muscle metabolism has not been investigated, and the contribution of skeletal muscle to the ROR؊/؊ phenotype has not been resolved. We utilize ectopic dominant negative ROR␣ expression in skeletal muscle cells to understand the regulatory role of RORs in this major mass peripheral tissue. Exogenous dominant negative ROR␣ expression in skeletal muscle cells represses the endogenous levels of ROR␣ and -␥ mRNAs and ROR-dependent gene expression. Moreover, we observed attenuated expression of many genes involved in lipid homeostasis. Furthermore, we show that the muscle carnitine palmitoyltransferase-1 and caveolin-3 promoters are directly regulated by ROR and coactivated by p300 and PGC-1. This study implicates RORs in the control of lipid homeostasis in skeletal muscle. In conclusion, we speculate that ROR agonists would increase fatty acid catabolism in muscle and suggest selective activators of ROR may have therapeutic utility in the treatment of obesity and atherosclerosis.Members of the nuclear hormone receptor (NR) 1 superfamily bind specific DNA elements and function as transcriptional regulators (1, 2). This group includes the "orphan NRs," which have no known ligands in the "classical sense." The orphan receptor, ROR/RZR (retinoic acid receptor-related orphan receptor), is closely related to Rev-erbA␣, RVR/Rev-erb␤/BD73, and the Drosophila orphan receptor, E75A, particularly in the DNA-binding domain and the putative ligand-binding domain. ROR, Rev-erbA␣, and RVR bind as monomers to an asymmetric (A/T) 6 RGGTCA motif. ROR functions as a constitutive transactivator of gene expression, and in contrast, Rev-erbA␣ and RVR do not activate transcription, mediate transcriptional repression, and can repress constitutive trans-activation from this motif by ROR␣ (3-9).Three ROR/RZR genes have been identified; ROR␣ encodes four ROR␣ isoforms ␣1, ␣2, ␣3, and RZR␣, which are alternatively spliced products of the ROR␣ gene and are predominantly expressed in blood, brain, skeletal muscle, and fat cells (8,10). ROR␤/RZR␤ is expressed specifically in the brain (11), and ROR␥ is found at high levels in skeletal muscle (12)(13)(14).Genetic studies have implicated ROR␣ in the ...

Understanding the interaction between broadly neutralizing antibodies and their epitopes provides a basis for the rational design of a preventive hepatitis C virus (HCV) vaccine. CBH-2, HC-11, and HC-1 are representatives of antibodies to overlapping epitopes on E2 that mediate neutralization by blocking virus binding to CD81. To obtain insights into escape mechanisms, infectious cell culture virus, 2a HCVcc, was propagated under increasing concentrations of a neutralizing antibody to isolate escape mutants. Three escape patterns were observed with these antibodies. First, CBH-2 escape mutants that contained mutations at D431G or A439E, which did not compromise viral fitness, were isolated. Second, under the selective pressure of HC-11, escape mutations progressed from a single L438F substitution at a low antibody concentration to double substitutions, L438F and N434D or L438F and T435A, at higher antibody concentrations. Escape from HC-11 was associated with a loss of viral fitness. An HCV pseudoparticle (HCVpp) containing the L438F mutation bound to CD81 half as efficiently as did wild-type (wt) HCVpp. Third, for HC-1, the antibody at a critical concentration completely suppressed viral replication and generated no escape mutants. Epitope mapping revealed contact residues for CBH-2 and HC-11 in two regions of the E2 glycoprotein, amino acids (aa) 425 to 443 and aa 529 to 535. Interestingly, contact residues for HC-1 were identified only in the region encompassing aa 529 to 535 and not in aa 425 to 443. Taken together, these findings point to a region of variability, aa 425 to 443, that is responsible primarily for viral escape from neutralization, with or without compromising viral fitness. Moreover, the region aa 529 to 535 is a core CD81 binding region that does not tolerate neutralization escape mutations.Up to 170 million people worldwide are chronically infected with hepatitis C virus (HCV), with many at significant risk for liver failure and hepatocellular carcinoma (http://www.who.int /vaccine_research/diseases/viral_cancers/en/index2.html). The virus is transmitted primarily by parenteral routes and injection drug use in developed countries, whereas contaminated injection equipment appears to be the major risk factor for HCV infection in developing countries. From unsafe needle injections alone, the World Health Organization estimates an annual increase in the global burden by 2 million new infections (35). Current therapy with combined pegylated interferon and ribavirin has led to clinical improvement for some patients, but treatment is associated with adverse side effects and a high relapse rate off therapy. Clearly, additional approaches are needed for treatment and prevention of infection. However, an effective HCV vaccine has yet to be achieved, despite considerable effort. A major impediment is the genetic diversity of the virus. The phylogenetic tree of HCV contains seven major genotypes with more than 30% divergence between genotypes, and each genotype contains a large number of related subtype...

The E2 envelope glycoprotein of hepatitis C virus (HCV) binds to the host entry factor CD81 and is the principal target for neutralizing antibodies (NAbs). Most NAbs recognize hypervariable region 1 on E2, which undergoes frequent mutation, thereby allowing the virus to evade neutralization. Consequently, there is great interest in NAbs that target conserved epitopes. One such NAb is AP33, a mouse monoclonal antibody that recognizes a conserved, linear epitope on E2 and potently neutralizes a broad range of HCV genotypes. In this study, the X-ray structure of AP33 Fab in complex with an epitope peptide spanning residues 412 to 423 of HCV E2 was determined to 1.8 Å. In the complex, the peptide adopts a ␤-hairpin conformation and docks into a deep binding pocket on the antibody. The major determinants of antibody recognition are E2 residues L413, N415, G418, and W420. The structure is compared to the recently described HCV1 Fab in complex with the same epitope. Interestingly, the antigen-binding sites of HCV1 and AP33 are completely different, whereas the peptide conformation is very similar in the two structures. Mutagenesis of the peptide-binding residues on AP33 confirmed that these residues are also critical for AP33 recognition of whole E2, confirming that the peptide-bound structure truly represents AP33 interaction with the intact glycoprotein. The slightly conformation-sensitive character of the AP33-E2 interaction was explored by cross-competition analysis and alaninescanning mutagenesis. The structural details of this neutralizing epitope provide a starting point for the design of an immunogen capable of eliciting AP33-like antibodies. Hepatitis C virus (HCV) infects an estimated 2 to 3% of the world population (4, 31) and is a major cause of chronic liver disease. The standard of care for chronic infection-a combination of pegylated alpha interferon and ribavirin-is effective in only 50% of patients infected with genotype 1 and is further limited by significant side effects, resistance, and high costs. This treatment has recently been updated to include two new directacting antivirals (DAAs), boceprevir (30) and telaprevir (36). A combination of either of these with pegylated alpha interferon and ribavirin has become the new standard therapy for patients with HCV genotype 1 infections. This approach to treatment, while improving the sustained virological response (SVR) rate compared to pegylated alpha interferon and ribavirin alone, still suffers a number of drawbacks: the regimen is restricted to patients with genotype 1 HCV infection, and there is an increased rate of adverse effects. Additionally, since the DAA treatment still requires coadministration of pegylated alpha interferon and ribavirin to reduce the risk of selecting for resistant strains (45), the problems of high cost and low tolerance associated with these drugs remain. There is therefore a pressing need to develop alternative anti-HCV therapies, particularly in the arena of preventative or therapeutic vaccines. The observation that some...

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.alanine scanning | immune recognition | HCV | clustering | hotspots H epatitis C virus (HCV) infects ∼185 million of the world's population, with 3-4 million new infections each year. Infection often leads to chronic hepatitis, cirrhosis, and hepatocellular carcinoma, and is a leading reason for liver transplantation (1). Despite recently developed direct-acting antiviral agents, there is a major need for a preventive HCV vaccine, because of the high cost of treatment therapies-which limit their clinical use-a high rate of asymptomatic and untreated infected individuals (over 95% of the infected population) (2, 3), concern of viral resistance to direct-acting antiviral agents (4), and that treatment-induced cure in patients with established cirrhosis does not eliminate the risk of hepatocellular carcinoma (5).A major obstacle to HCV vaccine development efforts is the extreme diversity of the virus and its high rate of mutation, which allows it to actively evade the immune response in infected individuals. Critical to the development of an effective vaccine is the identification and characterization of conserved epitopes associated with viral neutralization. The antibody response to HCV is directed primarily against the E2 glycoprotein because E2 directly interacts with the HCV coreceptors, scavenger receptor class B type 1 (SR-B1) (6) and the tetraspan...

Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCEFiloviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks. F iloviruses consisting of Marburg virus (MARV), Ravn virus (RAVV), and five species of ebolavirus, Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), Reston virus (RESTV), and Taï Forest virus (TAFV), are causative agents of severe hemorrhagic fever in humans and nonhuman primates (NHPs) (1, ...