Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Ebola virus (EBOV) and Marburg virus (MARV) of the virus familyFiloviridae are emerging and reemerging pathogens that cause hemorrhagic fever with high mortality rates in humans and nonhuman primates 1-3 . The public health concern about filoviruses has increased in recent years as a result of increased awareness and frequency of cases in central Africa as evidenced by the current outbreak of MARV in Angola 4 and also because filoviruses are considered to be potential agents of bioterrorism 5 . Currently, there are no EBOV or MARV vaccines or therapies approved for human use. Recently, we generated live attenuated recombinant vesicular stomatitis viruses (rVSV) expressing the transmembrane glycoprotein of Zaire ebolavirus (ZEBOV; VSV ∴∆G/ZEBOVGP) and MARV (VSV∆G/MARVGP) 6 . Our study evaluated the utility of these rVSV vectors as candidate vaccines for EBOV and MARV using the cynomolgus macaque model.Filovirus vaccine research has been extensively reviewed in the past and has primarily focused on EBOV 7,8 . The first EBOV vaccine to protect nonhuman primates was a DNA prime-adenovirus boost approach using both the glycoprotein and nucleoprotein as target antigens 9 . This approach required several months for immunity to develop, which limited the utility of this strategy. More recently, an accelerated vaccine was described. A single immunization of nonhuman primates with 2 × 10 12 particles of an equal mixture of human adenovirus 5 vectors carrying either the gene encoding ZEBOV glycoprotein or the gene encoding ZEBOV nucleoprotein resulted in complete protection against ZEBOV 10 . Despite the intriguing success of the adenovirus vaccine, preexisting immunity rates of between 40 and 60% have been reported to adenovirus in the human population and this may eventually limit the utility of this approach [11][12][13] .A smaller number of efforts have focused on developing vaccines against MARV. Alphavirus replicons expressing MARV proteins protected cynomolgus monkeys from homologous MARV challenge 14 . Subsequent studies evaluating this platform as a vaccine for EBOV were less encouraging, as the EBOV counterpart of this alphavirus replicon platform was unable to protect any animal against lethal EBOV challenge under similar test conditions 7 . The ideal vaccine would protect humans from infection from all four EBOV species (ZEBOV, Sudan ebolavirus (SEBOV), Reston ebolavirus, Ivory Coast ebolavirus) and MARV. Although the adenovirusbased vaccine platform has completely protected nonhuman primates against ZEBOV 9,10 , and the platform based on alphavirus replicons protected monkeys against MARV 14 , no platform has demonstrably protected nonhuman primates against both of these viruses.Vaccines based on live attenuated rVSV have been highly effective in animal models and are particularly attractive because they can ...

Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-␣Further, VP24 is found to specifically interact with karyopherin ␣1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin ␣2, ␣3, or ␣4. Overexpression of VP24 results in a loss of karyopherin ␣1-PY-STAT1 interaction, indicating that the VP24-karyopherin ␣1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.The filoviruses, Ebola virus (EBOV) and Marburg virus, cause periodic outbreaks of severe hemorrhagic fever in humans. In EBOV outbreaks consisting of more than 10 reported cases, mortality rates have ranged from 40 to 90% (41), and Marburg virus outbreaks have had reported case fatality rates ranging from 25 to 80% (13). This extreme virulence has made Ebola and Marburg viruses of concern both as naturally emerging pathogens and as potential bioweapons (41).The molecular mechanisms contributing to the severe pathogenesis of filovirus infection are poorly understood. Several potential mechanisms contributing to EBOV virulence have been reviewed (41). These include cytotoxicity of the viral glycoprotein, the production of proinflammatory cytokines, and the dysregulation of the coagulation cascade due to the production of tissue factor (14,20,21,62,64). Infection also appears to induce a general immune suppression (11, 53). Possible mechanisms contributing to this suppression include inhibition of dendritic cell activation and an induction of lymphocyte apoptosis (2,8,18,22,43). Each of these pathogenic processes likely occurs as a result of the active replication of the virus. Thus, the ability of the virus to counteract early antiviral responses, including those of the host's interferon system, likely plays an important role in EBOV virulence (41).EBOV encodes mechanisms to counteract the host interferon (IFN) response by blocking both production of IFN-␣/␤ and cellular responses to IFN-␣/␤ or -␥ treatment (6,24,26,27). We previously demonstrated that the EBOV VP35 protein suppresses IFN-␣/␤ production by inhibiting the activation of interferon regulatory factor 3 (IRF-3) (5, 7, 51), and subsequent studies confirm that VP35 exerts this function (8, 28). However, the manner in which EBOV blocks signaling from the IFN-␣/␤ or -␥ receptor has remained incompletely defined.IFN-␣/␤, a family of structurally related proteins, and IFN-␥ bind to two distinct receptors but activate similar signaling pathways (reviewed in reference 38). For both pathways, ligand binding activates receptor-associated Jak family tyrosine kinases. These undergo auto-and transphosphorylation and phosphorylate the cytoplasmic domains of the receptor subunits. The receptor-associated phosphotyrosine residues then serve as docking sites for the SH2 domains of STAT proteins. The receptor-associated STATs then undergo tyrosine-phosphorylation and form homo-or heterodimers via reciprocal SH2 domai...

In the present study, we have investigated processing and maturation of the envelope glycoprotein (GP) of Ebola virus. When GP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments, the mature form and two different precursors were identified. First, the endoplasmic reticulum form preGP er , full-length GP with oligomannosidic N-glycans, was detected. preGP er (110 kDa) was replaced by the Golgi-specific form preGP (160 kDa), fulllength GP containing mature carbohydrates. preGP was finally converted by proteolysis into mature GP 1,2 , which consisted of two disulfide-linked cleavage products, the aminoterminal 140-kDa fragment GP 1 , and the carboxyl-terminal 26-kDa fragment GP 2 . GP 1,2 was also identified in Ebola virions. Studies employing site-directed mutagenesis revealed that GP was cleaved at a multibasic amino acid motif located at positions 497 to 501 of the ORF. Cleavage was blocked by a peptidyl chloromethylketone containing such a motif. GP is cleaved by the proprotein convertase furin. This was indicated by the observation that cleavage did not occur when GP was expressed in furin-defective LoVo cells but that it was restored in these cells by vector-expressed furin. The Reston subtype, which differs from all other Ebola viruses by its low human pathogenicity, has a reduced cleavability due to a mutation at the cleavage site. As a result of these observations, it should now be considered that proteolytic processing of GP may be an important determinant for the pathogenicity of Ebola virus.Ebola and Marburg viruses, the two species within the family Filoviridae (1), are among the most pathogenic agents causing fulminant hemorrhagic fever in humans and nonhuman primates. Yet, we are only beginning to understand the interactions of these viruses with their hosts, and our knowledge on genetics, pathogenicity, and natural history is still limited. Although outbreaks have so far always been self-limiting, our ignorance concerning the natural reservoir and the lack of immunoprophylactic and chemotherapeutic measures makes these infections a matter of high concern in biomedical science. The chronology of human epidemics and epizootics in nonhuman primates proves that filoviruses are prototypes of emerging͞re-emerging pathogens. The recent emergence of a new Ebola subtype in Cote d'Ivoire (2) and the re-emergence of Ebola subtype Zaire in former Zaire (3) and Gabon (4, 5) once again showed that these viruses have altered from exotic agents to pathogens of serious public health concerns.The genomes of filoviruses display linear arranged genes on a single negative-stranded RNA molecule that encode the seven structural proteins in the order nucleoprotein, virion structural protein (VP) 35, VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L) (6-8). In general, filoviral genes are transcribed into monocistronic subgenomic RNA species (mRNA) (6, 9). In contrast to all other filoviral genes, including the GP gene of Marburg virus (10), the organization and tran...

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