ADV−GP ADV−GP consists of an adenoviral (ADV) vector encoding the Ebola glycoprotein (GP) [Ref77:Sullivan et al., 2003]. To make ADV-GP, the BamHI/EcoRI fragment of GP(Z) was digested from PGEM-3Zf(-)-GP, treated with Klenow, and inserted into HindIII/XbaI/Kle/CIP-treated pRc/CMV plasmid. The resulting plasmid was digested by NruI/DraIII and treated with Klenow. The NruI/DraIII/Kle fragment containing the CMV enhancer, GP(Z) DNA and bovine growth hormone polyadenylation signal was inserted into the BgIII site of the adenoviral shuttle plasmid pAdBgIII26. The adenovirus, a first generation dl 309-based Ad5 vector, contained a deletion in E1 to render the vector replication defective and a partial deletion/substitution in E3, which disrupts the coding sequences for the E3 proteins with a relative molecular mass of 14,700, 14,500 and 10,400, respectively [Ref79: Sullivan et al., 2000]. GP This vaccine comes from RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein [Ref86: Geisbert et al., 2002]. The EBOV GP gene was inserted into a VACV transfer vector plasmid, and recombinant VACV expressing EBOV GP were isolated [Ref86:Geisbert et al., 2002]. GP and NP The vaccine components include nucleoprotein and three subtypes of Ebola glycoprotein, Zaire, Ivory Coast and Sudan (GP(Z,IC,S) + NP) [Ref79:Sullivan et al., 2000]. GP and NP This immunogen is composed of RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein [Ref86:Geisbert et al., 2002]. The Ebola NP and GP genes from the Mayinga strain of Ebola virus were derived from pSP64- and pGEM3Zf(-)-based plasmids. The BamHI±EcoRI (2.3 kb) and BamHI±KpnI (2.4 kb) fragments containing the NP and GP genes, respectively, were subcloned into a shuttle vector digested with BamHI and EcoRI within a polylinker sequence flanked by ClaI sites. For cloning of the GP gene, overhanging ends produced by KpnI (in the GP fragment) and EcoRI (in the shuttle vector) were made blunt by incubation with T4 DNA polymerase. From the shuttle vector, NP or GP genes were transferred as ClaI-fragments into the ClaI site of the replicon clone, resulting in plasmids encoding the NP or GP gene in place of the VEE structural protein genes [Ref89:Pushko et al., 2000]. NP NP (nucleoprotein) is a structural gene product of the Ebola virus [Ref103:Xu et al., 1998]. Plasmids containing the sGP cDNAs are used to subclone the relevant inserts into CMV expression vectors, which utilize the bovine growth hormone polyadenylation sequence. The plasmid pSP64-NP2 is digested with EcoRI/BamHI, and treated with Klenow enzyme. The NP insert is cloned into CMV treated with BamHI, Klenow enzyme, followed by heat inactivation and Bg/II digestion [Ref103:Xu et al., 1998]. VRP expressing VP24 VP24 is an Ebola virus protein. It is membrane associated and is most likely located on the inside of the membrane. The function of VP24 is not known but it may serve as a minor matrix protein, facilitating the interaction of VP40 and/or GP with the RNP complex, or function in the uncoating of the virion during infection [Ref88:Wilson et al., 2001]. Replicon RNAs were packaged into particles. Briefly, capped replicon RNAs were produced in vitro by T7 runoff transcription of NotI-digested plasmid templates using the RiboMAX T7 RNA polymerase kit. BHK cells were cotransfected with the replicon RNAs and two RNAs expressing the VEE virus structural proteins. The cell culture supernatants were harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by centrifugation through a 20% sucrose cushion. Packaged VRPs were suspended in phosphatebuffered saline and titers were determined as immunofluorescent foci after infection of Vero cells as described using either EBOV-immune rabbit serum or mouse monoclonal antibodies to VP24 (Z-AC01-BG11-01), VP35 (M-HC01-AF11), or VP40 (M-HD06-AD10) [Ref88:Wilson et al., 2001]. VRP expressing VP30 VP30 is an Ebola virus protein. It associates with the genomic RNA in a ribonucleoprotein complex. The VP30 protein is not essential for replication, but it is necessary for efficient transcription in this system. It has also recently been shown to be essential for the recovery of infectious EBOV-Z from cloned cDNAs [Ref88:Wilson et al., 2001]. Replicon RNAs were packaged into particles. Briefly, capped replicon RNAs were produced in vitro by T7 runoff transcription of NotI-digested plasmid templates using the RiboMAX T7 RNA polymerase kit. BHK cells were cotransfected with the replicon RNAs and two RNAs expressing the VEE virus structural proteins. The cell culture supernatants were harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by centrifugation through a 20% sucrose cushion. Packaged VRPs were suspended in phosphatebuffered saline and titers were determined as immunofluorescent foci after infection of Vero cells as described using either EBOV-immune rabbit serum or mouse monoclonal antibodies to VP24 (Z-AC01-BG11-01), VP35 (M-HC01-AF11), or VP40 (M-HD06-AD10) [Ref88:Wilson et al., 2001]. VRP expressing VP35 VP35 is an Ebola virus protein. It associates with the genomic RNA in a ribonucleoprotein complex. It is essential for replication and encapsidation of the EBOV genome. The VP35 protein has also recently been shown to be essential for the recovery of infectious EBOV-Z from cloned cDNAs. In addition to being an essential component of the replication complex, VP35 was also recently implicated as an interferon antagonist. VP35 may therefore facilitate viral replication in infected cells by blocking the induction of antiviral immune responses normally induced by the production of interferon [Ref88:Wilson et al., 2001]. Replicon RNAs were packaged into particles. Briefly, capped replicon RNAs were produced in vitro by T7 runoff transcription of NotI-digested plasmid templates using the RiboMAX T7 RNA polymerase kit. BHK cells were cotransfected with the replicon RNAs and two RNAs expressing the VEE virus structural proteins. The cell culture supernatants were harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by centrifugation through a 20% sucrose cushion. Packaged VRPs were suspended in phosphatebuffered saline and titers were determined as immunofluorescent foci after infection of Vero cells as described using either EBOV-immune rabbit serum or mouse monoclonal antibodies to VP24 (Z-AC01-BG11-01), VP35 (M-HC01-AF11), or VP40 (M-HD06-AD10) [Ref88:Wilson et al., 2001]. VRP expressing VP40 VP40 is an Ebola virus protein. It is membrane-associated and is most likely located on the inside of the membrane. VP40 has been shown to associate with cell membranes, where it is believed to be involved in maturation of the virus by inducing viral assembly at the plasma membrane of infected cells [Ref88:Wilson et al., 2001]. Replicon RNAs were packaged into particles. Briefly, capped replicon RNAs were produced in vitro by T7 runoff transcription of NotI-digested plasmid templates using the RiboMAX T7 RNA polymerase kit. BHK cells were cotransfected with the replicon RNAs and two RNAs expressing the VEE virus structural proteins. The cell culture supernatants were harvested approximately 30 h after transfection and the replicon particles were concentrated and partially purified by centrifugation through a 20% sucrose cushion. Packaged VRPs were suspended in phosphatebuffered saline and titers were determined as immunofluorescent foci after infection of Vero cells as described using either EBOV-immune rabbit serum or mouse monoclonal antibodies to VP40 (M-HD06-AD10) [Ref88:Wilson et al., 2001]. GP-VRP The Ebola GP genes from the Mayinga strain of Ebola virus were derived from pGEM3Zf(-)-based plasmid. The BamHI±KpnI (2.4 kb) fragment containing the GP gene was subcloned into a shuttle vector. From the shuttle vector, GP gene was transferred as ClaI-fragment into the ClaI site of the replicon clone, resulting in plasmids encoding the GP gene in place of the VEE structural protein genes [Ref89:Pushko et al., 2000]. DNA vaccine expressing sGP sGP is a secreted or transmembrane form of glycoprotein [Ref103:Xu et al., 1998]. Plasmids containing the sGP cDNAs are used to subclone the relevant inserts into CMV expression vectors, which utilize the bovine growth hormone polyadenylation sequence. The plasmid pCRII-sGP is digested with EcoRI and treated with Klenow enzyme, and the resulting fragment is inserted into the BamHI/Bg/II CMV plasmid, which has been incubated with Klenow fragment and calf intestinal phosphate (CIP), and phenol chloroform extracted [Ref103:Xu et al., 1998]. NP-VRP The Ebola NP gene from the Mayinga strain of Ebola virus were derived from pSP64-based plasmid. The BamHI±EcoRI (2.3 kb) fragment containing the NPgene, was subcloned into a shuttle vector digested with BamHI and EcoRI within a polylinker sequence flanked by ClaI sites. From the shuttle vector, NP gene was transferred as ClaI-fragments into the ClaI site of the replicon clone, resulting in plasmids encoding the NP gene in place of the VEE structural protein genes [Ref89:Pushko et al., 2000]. Ebola virus DNA vaccine encoding ZEBOV GP and SEBOV GP