ABSTRACT Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.

Abstract Multiple Sclerosis (MS) is a neurological disorder characterized by the pathological trafficking of autoreactive leukocytes into the central nervous system (CNS). We previously showed that removal of CXCL12 from abluminal surfaces of the CNS microvasculature facilitates the entry of CXCR4-expressing leukocytes within MS tissues and in mice with experimental autoimmune encephalomyelitis (EAE), a model for MS. Recently, the orphan receptor CXCR7 was determined to bind CXCL12/11. Evidence suggests that CXCR7 mainly regulates CXCR4 signaling by sequestering CXCL12 from extracellular milieu. Using MS and EAE specimens, we tested the hypothesis that inhibition of CXCR7 would prevent leukocyte migration into the CNS and induction of autoimmunity. CXCR7 is expressed by both human and murine CNS endothelium and CXCR7 antagonism completely blocks CXCL12 internalization in brain microvascular cells in vitro. In vivo antagonism of CXCR7 led to dose-dependent inhibition of EAE and ameliorated clinical symptoms in mice with ongoing disease. Immunohistochemical analysis revealed increased leukocytes within meningeal vessels, decreased perivascular infiltrates and parenchymal entry of leukocytes compared with controls. Abluminal CXCL12 expression persisted only in mice treated with high doses of CXCR7 antagonist. The benefits of persistent abluminal CXCL12 location during EAE suggest that pharmacologic targeting of CXCR7 may have therapeutic utility for the treatment of MS.

Rift Valley fever virus (RVFV) has been cited as a potential biological-weapon threat due to the serious and fatal disease it causes in humans and animals and the fact that this mosquito-borne virus can be lethal in an aerosolized form. Current human and veterinary vaccines against RVFV, however, are outdated, inefficient, and unsafe. We have incorporated the RVFV glycoprotein genes into a nonreplicating complex adenovirus (CAdVax) vector platform to develop a novel RVFV vaccine. Mice vaccinated with the CAdVax-based vaccine produced potent humoral immune responses and were protected against lethal RVFV infection. Additionally, protection was elicited in mice despite preexisting immunity to the adenovirus vector.

Traditional vaccine development platforms such as live-attenuated virus, killed virus, or recombinant subunit-based vaccines are often effective in eliciting long-term immunity to a number of infectious human pathogens. However, for many human pathogens, vaccine platforms such as these are unsuitable for human use due to safety concerns, poor efficacy, or simple impracticality. As a result, much work has focused on the use of recombinant virus vectors as a means for vaccination against human pathogens. Viral vectors can express foreign proteins at high levels in host cells, resulting in strong, long-lasting immune responses against the target proteins. This chapter describes the use of virus vectors in the context of vaccination against human pathogens. Various vector platforms are discussed, compared, and contrasted.

ABSTRACT Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes.

During the last decade, sphingolipid deregulation, namely the balance between the pro-apoptotic molecule ceramide and the anti-apoptotic sphingolipid sphingosine-1-phosphate, has emerged as an important factor in cancer pathology and resistance to therapy. Thus, our research has been focused on developing drugs that are able to restore normal sphingolipid balance, precisely through increasing the levels of ceramide and decreasing sphingosine-1-phosphate. Particularly, inhibition of the ceramide metabolizing enzyme acid ceramidase, whose over-expression in cancer cells has been implicated in resistance to treatment, is proving to be an efficient and promising strategy. In this review, we consider our recent work with acid ceramidase inhibitors, in combination with radiation or gene therapy as a sensitizer that enhance cancer therapy.

ABSTRACT Filoviruses (Ebola and Marburg viruses) are among the deadliest viruses known to mankind, with mortality rates nearing 90%. These pathogens are highly infectious through contact with infected body fluids and can be easily aerosolized. Additionally, there are currently no licensed vaccines available to prevent filovirus outbreaks. Their high mortality rates and infectious capabilities when aerosolized and the lack of licensed vaccines available to prevent such infectious make Ebola and Marburg viruses serious bioterrorism threats, placing them both on the category A list of bioterrorism agents. Here we describe a panfilovirus vaccine based on a complex adenovirus (CAdVax) technology that expresses multiple antigens from five different filoviruses de novo. Vaccination of nonhuman primates demonstrated 100% protection against infection by two species of Ebola virus and three Marburg virus subtypes, each administered at 1,000 times the lethal dose. This study indicates the feasibility of vaccination against all current filovirus threats in the event of natural hemorrhagic fever outbreak or biological attack.

There are legitimate concerns that the highly pathogenic H5N1 avian influenza virus could adapt for human-to-human transmission and cause a pandemic similar to the 1918 "Spanish flu" that killed 50 million people worldwide. We have developed pandemic influenza vaccines by incorporating multiple antigens from both avian and Spanish influenza viruses into complex recombinant adenovirus vectors. In vaccinated mice, these vaccines induced strong humoral and cellular immune responses against pandemic influenza virus antigens, and protected vaccinated mice against lethal H5N1 virus challenge. These results indicate that this multi-antigen, broadly protective vaccine may serve as a safer and more effective approach than traditional methods for development of a pandemic influenza vaccine.

PURPOSE:Alterations in ceramide metabolism have been reported in prostate cancer (PCa), resulting in escape of cancer cells from ceramide-induced apoptosis. Specifically, increased expression of lysosomal acid ceramidase (AC) has been shown in some primary PCa tissues and in several PCa cell lines. To determine if this represents a novel therapeutic target, we designed and synthesized LCL204, a lysosomotropic analog of B13, a previously reported inhibitor of AC METHODS: Prostate cancer cell lines were treated with LCL204 for varying times and concentrations. Effects of treatment on cytotoxicity, sphingolipid content, and apoptotic markers were assessed.RESULTS:Treatment of DU145 PCa cells resulted in increased ceramide and decreased sphingosine levels. Interestingly, LCL204 caused degradation of AC in a cathepsin-dependent manner. We also observed rapid destabilization of lysosomes and the release of lysosomal proteases into the cytosol following treatment with LCL204. Combined, these events resulted in mitochondria depolarization and executioner caspase activation, ultimately ending in apoptosisCONCLUSIONS:These results provide evidence that treatment with molecules such as LCL204, which restore ceramide levels in PCa cells may serve as a new viable treatment option for PCa.

There are approximately 100 million new cases of dengue (DEN) virus infection each year. Infection can result in illness ranging from a mild fever to hemorrhaging, shock, or even death. There are four serotypes of dengue virus (DEN1-4), and immunity to one serotype does not cross protect from infection with other serotypes. Currently there are no approved vaccines for dengue fever. In this report, we describe the construction of a bivalent dengue virus vaccine using a complex recombinant adenovirus approach to express multiple genes of DEN1 and DEN2 serotypes. In vaccinated mice, this vector induced humoral immune responses against all four dengue serotypes as measured by enzyme-linked immunosorbent assay. However, the neutralizing antibody responses were specific for DEN1 and DEN2 serotypes. Expansion of this vaccine development platform towards the DEN3 and DEN4 serotypes can lead towards the development of an adenovirus-based tetravalent dengue vaccine.

ABSTRACT West Nile Virus (WNV), a member of the family Flaviviridae, was first identified in Africa in 1937. In recent years, it has spread into Europe and North America. The clinical manifestations of WNV infection range from mild febrile symptoms to fatal encephalitis. Two genetic lineages (lineages I and II) are recognized; lineage II is associated with mild disease, while lineage I has been associated with severe disease, including encephalitis. WNV has now spread across North America, significantly affecting both public and veterinary health. In the efforts to develop an effective vaccine against all genetic variants of WNV, we have studied the feasibility of inducing both neutralizing and cellular immune responses by de novo synthesis of WNV antigens using a complex adenoviral vaccine (CAdVax) vector. By expressing multiple WNV proteins from a single vaccine vector, we were able to induce both humoral and cellular immune responses in vaccinated mice. Neutralization assays demonstrated that the antibodies were broadly neutralizing against both lineages of WNV, with a significant preference for the homologous lineage II virus. The results from this study show that multiple antigens synthesized de novo from a CAdVax vector are capable of inducing both humoral and cellular immune responses against WNV and that a multiantigen approach may provide broad protection against multiple genetic variants of WNV.

Head and neck squamous cell cancers (HNSCC) are particularly aggressive and are resistant to many forms of treatment. Ceramide metabolism has been shown to play an important role in cancer progression and cancer resistance to therapy in many tumor models, including HNSCC. Here, we study the role of the ceramide-metabolizing enzyme acid ceramidase (AC) in therapeutic responses in HNSCC. First, we show that AC is over-expressed in 70% of head and neck squamous cell tumors compared with normal tissues, suggesting that this enzyme may play an important role in facilitating HNSCC growth. Next, comparison of three HNSCC cell lines with low, medium, and high levels of AC reveals an inverse correlation between the levels of AC and their response to exogenous C-6-ceramide. Furthermore, over-expression of AC in SCC-1 cells increased resistance to Fas-induced cell killing. Conversely, down-regulation of AC using specific AC small interfering RNA (siRNA) sensitized the SCC-1 cancer cell line to Fas-induced apoptosis. Finally, we show that the AC inhibitor LCL 204 can sensitize HNSCC cell lines to Fas-induced apoptosis both in vitro and in a xenograft model in vivo, suggesting that the combination of FasL gene therapy and LCL 204 may become a new treatment option for advanced-stage head and neck cancer.

Treatment of different cancer cell lines with desipramine induced a time- and dose-dependent downregulation of acid ceramidase. Desipramine’s effect on acid ceramidase appeared specific for amphiphilic agents (desipramine, chlorpromazine, and chloroquine) but not other lysomotropic agents such as ammonium chloride and bafilomycin A1, and was not transcriptionally regulated. The cathepsin B/L inhibitor, CA074ME, but not the cathepsin D inhibitor, pepstatin A, blocked desipramine’s effect on acid ceramidase. Desipramine led to a more pronounced downregulation of sphingosine compared to ceramide suggesting acid ceramidase inhibition is important to desipramine’s mechanism of action. This study reveals a new mechanism of action for desipramine.

INTRODUCTION AND OBJECTIVE: RCC is the sixth leading cause of cancer death in USA. One third of patients present with metastatic disease. Current systemic therapies have limited results. Gene therapy including pro-apoptotic gene therapy is a promising tool under investigation for treatment of RCC. However, inability to deliver lethal genes to all tumor cells is a problem. FasL is a pro- apoptotic gene that is a member of the Tumor Necrosis Factor family. Although ACHN cells were resistant to convention Fas monoclonal Ab (CH-11), we were able to induce apoptosis by AdGFPFasLTET gene therapy. Ceramide generation was observed after treatment. Ceramide, the basic unit of sphingolipid, is involved in cell cycle arrest and/ or apoptosis. On Using Ceramide or ceramide analogue, we were able to sensitize ACHN cells to CH-11. This indicates that ceramide plays a significant role in sensitizing ACHN to gene therapy. METHODS: ACHN treated with CH-11, AdGFPFasLTETTET, or AdGFP. AdGFPFasLTETTET is replication-deficient adenovirus containing a modified murine Fas ligand gene fused to green fluorescent protein (GFP), with a promoter can down-regulate gene expression in presence of doxycycline. AdGFP that contain the GFP gene alone was used as a control. Viability of the cells was assessed using MTS assay. Cells were also evaluated for apoptosis by alteration of mitochondrial membrane potential (MMP) by FACS and caspase 3/7 assay. Ceramide level with AdGFPFasLTET treatment was measured by HPLC-Ms in Lipidomics core . C6-De erythro-ceramide and lysosomotropic ceramide analogue LCL-385 were combined with CH-11 and viability was assessed by MTS. Apoptosis was confirmed using casapase 3/7 assay. RESULTS: ACHN cells were resistant to apoptosis when treated with CH-11. However, ACHN were extremely sensitive to AdGFPFasLTET while minimal cytotoxicity was seen with control virus AdGFP at the same MOI. Caspase 3/7 was increased by treatment with AdGFPFasLTET and not with AdGFP or CH-11. Increased ceramide level was observed with treatment with AdGFPFasLTET. C6-De-Erythro ceramide or LCL-385 were able to sensitize ACHN cells to CH-11 as assessed by MTS and casapse 3/7 assay. CONCLUSIONS: ACHN cells are extremely sensitive to AdGFPFasLTET treatment and resistant to CH-11. AdGFPFasLTET treatment results in Ceramide generation. Treating ACHN with Ceramide or ceramide analogue lead to sensitizing these cells to CH-11. These findings support the significance to investigate further role of ceramide in sensitizing resistant cancer cells to gene therapy or other chemotherapy.

It is increasingly apparent that sphingolipids, and in particular ceramide, are important mediators of intracellular response to stress from various stimuli. Many tumors have up-regulated enzymes such as the lysosomal enzyme acid ceramidase (AC) which deacylates ceramide forming sphingosine. This is presumed to allow the cell to survive during these times of stress. In a cancer this would likely be a negative event for the patient. The chicken anemia viral protein, Apoptin, causes tumor selective apoptosis in human tumor and transformed cells. Its mechanism of action is unknown but in a previous study, we demonstrated that ceramide elevation appears to contribute to Apoptin's mechanism of action. In the same study, reduced sensitivity to Apoptin was also observed in cells over-expressing AC, suggesting ceramide deacylation might serve to negate Apoptin induced apoptosis. We have now evaluated this further using PC3 prostate cancer cells, transfected with small interfering RNA (siRNA) targeted to AC. Using Western blotting, we observed 85% selective downregulation of AC by 48 hours post-transfection. This treatment alone resulted in inhibition of PC3 cell growth, with a 12% increased level of death rate. Mass Spectrometry demonstrated an increased ceramide level in almost all ceramide species examined and a significant decline in sphingosine concentration at 24 hours in siRNA-treated cells. Treatment with siRNAs followed by administration of Ad- GFPApoptin further reduced the number of viable cells, when compared to either of the treatments alone. In conclusion, downregulation of acid ceramidase by RNAi enhances the effects of Apoptin. Our study strongly suggests that the ceramide-acid ceramidase pathway represents a new target for the development of AC inhibitors to sensitize tumor cells to therapeutic agents.

ABSTRACT Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.

The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 muM) attenuated (30%; P<0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P<0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.