RECOMBINANT BIOLOGICALLY CONTAINED FILOVIRUS

Cross-Reference to Related Applications

The present application claims the benefit of the filing date of U.S. application Serial No. 61/019,532, filed on January 7, 2008, the disclosure of which is incorporated by reference herein.

Statement of Government Rights

The invention was made at least in part with a grant from the Government of the United States of America (Grant AI057153 from the National Institutes of Health). The Government has certain rights in the invention.

Background

Ebolaviruses cause hemorrhagic fevers in humans and nonhuman primates, with case fatality rates of 90% in some outbreaks (Sanchez et al., 2007). Ebolaviruses and the closely related Marburgviruses belong to the Filoviridae family (Feldman et al., 2004). Currently, there are no approved vaccines or antivirals for use against filoviruses, making biosafety level-4 (BSL- 4) containment a mandatory requirement for work with these viruses. The lack of sufficient BSL-4 space, trained personnel, and the rigors of working in BSL-4 laboratories have severely hampered basic research with Ebolaviruses as well as the development of vaccines and large-scale screening for effective antiviral compounds. These limitations have prompted examination of various steps in the Ebolavirus viral life cycle in the absence of infectious virus: (J) replication and transcription were studied by use of reporter gene assays that are based on the expression of necessary viral components from plasmids (Boehmann et al., 2005; Groseth et al., 2005; Muhlberger et al., 1999; Modrof et al., 2003; Modrof et al., 2002); (U) entry and fusion processes were assessed with pseudotyping assays that rely on the use of recombinant vesicular stomatitis or retroviruses (Yonezawa et al., 2005; Wool-Lewis et al., 1998; Takada et al., 1997; Marzi et al., 2006); and (m) budding was examined using virus-like particles that are generated from viral proteins provided by protein expression plasmids

(Jasenosky et al., 2001; Licata et al., 2004; Noda et al., 2002; McCarthy et al., 2006; Johnson et al., 2006). However, several recent findings suggest that data obtained with these artificial systems may not always be reproducible with live, authentic Ebolavirus (Neumann et al., 2005). Thus, biologically contained Ebolaviruses that resemble wild-type virus but can be handled outside BSL-4 containment are clearly needed.

Summary of the Invention

The invention provides a vaccine comprising an effective amount of a recombinant negative-sense, single stranded RNA virus, the genome of which contains a deletion of viral sequences corresponding to those for a nonstructural or nonglycosylated viral protein that is essential in trans for viral replication. In one embodiment, the deletion is effective to inhibit or prevent viral replication upon infection of a cell with the recombinant negative-sense, single stranded RNA virus. For example, the deletion may be effective to prevent expression of a functional nonstructural or nonglycosylated protein upon infection of a cell with the recombinant negative-sense, single stranded RNA virus. In one embodiment, the deletion may be in filovirus sequences for a viral protein corresponding to Ebola virus VP30 protein. In one embodiment, the deletion may be in filovirus sequences for a viral protein corresponding to Ebola virus L protein (polymerase), e.g., a polymerase that is filovirus subtype specific, such as one specific for the Zaire, Sudan, Cote d'lvoire, Bundibugyo or Reston subtype. The deletion in viral sequences of a negative-sense, single stranded RNA virus may include a deletion of 1 or more nucleotides, e.g., a deletion of at least 0.1%, 1%, 5%, 10%, 50%, 60%, 70%, 80%, 90%, or any integer in between, and up to 100% of the viral sequences corresponding to those for a nonstructural or nonglycosylated viral protein that is essential in trans for viral replication, e.g., sequences that do not overlap with those for another viral protein encoded by the viral genome. The deletion is one that is stable over multiple passages and is readily detectable, e.g., by RT-PCR. In one embodiment, the genome of the recombinant virus has a deletion in viral sequences for two or more nonstructural or nonglycosylated proteins, for example, a deletion in sequences for viral proteins that are not contiguous with each other, such as sequences for a viral protein corresponding to Ebola virus

VP30 protein and for a viral protein corresponding to Ebola virus L protein. In one embodiment, where the genome of the recombinant virus has a deletion in viral sequences for a nonstructural or nonglycosylated protein, at least a portion of the deleted viral sequences may be replaced with a detectable marker gene, e.g., a neo, gfp or luc gene, or a combination thereof. In one embodiment, where the genome of the recombinant virus has a deletion in viral sequences for two or more nonstructural and/or nonglycosylated proteins, at least a portion of one of the deleted viral sequences may be replaced with a detectable marker gene, e.g., a neo, gfp or luc gene, or a combination thereof. The vaccine of the invention may provide for subtype cross protection or for filovirus cross protection. In one embodiment, the invention provides isolated recombinant, biologically contained filovirus such as Ebola virus, the genome of which contains a deletion in sequences, e.g., those corresponding to Ebola virus VP30 sequences or to Ebola virus L sequences, or both. The deletion(s) is/are effective to inhibit or prevent viral replication, e.g., by preventing expression of a functional protein corresponding to Ebola virus VP30 protein or to Ebola virus L protein, or both, upon infection of a cell that lacks sequences that encode the functional protein (e.g., the cell that does not express functional VP30 and/or L in trans) with the recombinant, biologically contained filovirus, e.g., Ebola virus. In one embodiment, such an isolated virus is useful as a therapeutic vaccine, hi one embodiment, such an isolated virus is useful as a prophylactic vaccine. In one embodiment, at least 90% of sequences corresponding to Ebola virus VP30 sequences in the viral genome of the virus are deleted. In one embodiment, at least 90% of sequences corresponding to Ebola virus L sequences in the viral genome of the virus are deleted, hi one embodiment, at least 90% of sequences corresponding to Ebola virus VP30 and to L sequences in the viral genome of the virus are deleted. In one embodiment, the genome of the recombinant, biologically contained filovirus further comprises heterologous sequences, for instance, positioned within the deletion. The heterologous sequences may be selected as ones that are not toxic to one or more host cells, e.g., reporter, selectable marker or viral sequences (for instance, neo ^R , green fluorescent protein (GFP), luciferase or influenza virus sequences for mammalian cells). hi one embodiment, the invention provides isolated recombinant, biologically contained filovirus such as Ebola virus, the genome of which

contains a deletion in sequences corresponding to Ebola virus VP30 sequences. The deletion is effective to inhibit or prevent viral replication, e.g., by preventing expression of a functional protein corresponding to Ebola virus VP30 protein, upon infection of a cell that lacks sequences that encode the functional protein (e.g., the cell that does not express functional VP30 in trans) with the recombinant, biologically contained Ebola virus. In one embodiment, such an isolated virus is useful as a vaccine, hi one embodiment, at least 90% of sequences corresponding to Ebola virus VP30 sequences in the viral genome of the virus are deleted, hi one embodiment, the genome of the recombinant, biologically contained filovirus further comprises heterologous sequences, for instance, positioned within the deletion. The heterologous sequences may be selected as ones that are not toxic to one or more host cells, e.g., reporter, selectable marker or viral sequences, e.g., heterologous viral sequences. In one embodiment, the invention provides isolated recombinant, biologically contained filovirus such as Ebola virus, the genome of which contains a deletion in sequences corresponding to Ebola virus L protein sequences. The deletion is effective to inhibit or prevent viral replication, e.g., by preventing expression of a functional protein corresponding to Ebola virus L protein, upon infection of a cell that lacks sequences that encode the functional protein (e.g., the cell that does not express functional L protein in trans) with the recombinant, biologically contained Ebola virus. In one embodiment, such an isolated virus is useful as a vaccine. In one embodiment, at least 90% of sequences corresponding to Ebola virus L protein sequences in the viral genome of the virus are deleted. In one embodiment, the genome of the recombinant, biologically contained filovirus further comprises heterologous sequences, for instance, positioned within the deletion. The heterologous sequences may be selected as ones that are not toxic to one or more host cells, e.g., reporter, selectable marker or viral sequences, e.g., heterologous viral sequences. In one embodiment, the invention provides isolated recombinant, biologically contained filovirus such as Ebola virus, the genome of which contains deletions in sequences for two different filovirus proteins, such as those corresponding to Ebola virus VP30 and L protein sequences, or other combinations of nonstructural viral proteins. The deletions are effective to inhibit or prevent viral replication, e.g., by preventing expression of functional

proteins corresponding to Ebola virus VP30 and L proteins, or other combinations of nonstructural proteins, upon infection of a cell that lacks sequences that encode the functional proteins with the recombinant, biologically contained Ebola virus. In one embodiment, such an isolated virus is useful as a vaccine. In one embodiment, at least 90% of sequences corresponding to VP30 and L protein sequences in the viral genome of the virus are deleted. In one embodiment, the genome of the recombinant, biologically contained fϊlovirus further comprises heterologous sequences, for instance, positioned within the deletion. The heterologous sequences may be selected as ones that are not toxic to one or more host cells, e.g., reporter, selectable marker or viral sequences, such as heterologous viral sequences.

To prepare such viruses, a reverse genetics systems for negative-sense RNA viruses was exploited to generate Ebolaviruses that lack a substantial portion of the VP30 gene (which encodes an essential transcription factor), termed EbolaδVP30 virus, lack a substantial portion of the L gene, or lack a substantial portion of both genes. EbolaδVP30 viruses were maintained, genetically stable, and biologically confined to a cell line expressing VP30. Hence, the EbolaδVP30 virus fulfills several criteria of a vaccine virus: it can be grown to reasonably high titers in helper cells, is genetically stable (as determined by sequence analysis after seven serial passages in VP30-expressing Vero cells), and is safe. Moreover, as described herein, the resultant viruses resemble wild-type virus in their life cycle, their morphology, and their growth properties, but could be handled in a non-BSL-4 laboratory, opening new opportunities for study of the Ebolavirus life cycle and for the identification of effective antiviral compounds.

Other negative-sense, single stranded RNA viruses may likewise be manipulated, e.g., the genome of Nipah virus, Hendravirus, Henipavirus, and the like, may be manipulated to mutate or delete sequences corresponding to those for a nonstructural or nonglycoslyated viral protein that is required for viral replication. Thus, genomes of viruses in the following families may be manipulated to provide for an infectious, biologically contained virus that resembles wild-type virus in its life cycle, morphology, and growth properties, can be grown to reasonably high titers in helper cells, is genetically stable, and is safe: Bornaviridae, Rhabdoviridae, Filoviridae (genera Marburgvirus and

Ebolavirus), Paramyxoviridae, Avulavirus, Henipavirus, Morbillivirus, Respirovirus, or Rubulavirus.

As also described herein, immunized mice with EbolaδVP30 were protected from a lethal infection of mouse-adapted Ebola virus. The virus titers in the blood of immunized mice were more than 1000-fold lower than those in mock-immunized animals. Protection of mice vaccinated with EbolaδVP30 was associated with a high antibody response to the Ebola virus glycoprotein (GP) and the generation of an Ebola virus NP-specific CD8 ⁺ T-cell response. This demonstrates the potential of the EbolaδVP30 virus as a vaccine candidate. Thus, in one embodiment, the invention provides a vaccine comprising an effective amount of a recombinant filovirus, the genome of which contains a deletion of sequences corresponding to Ebola virus VP30, wherein the deletion is effective to prevent expression of a functional protein corresponding to Ebola virus VP30 upon infection of a cell that lacks sequences that encode a functional VP30 with the recombinant virus.

The invention also provides a method to prepare an infectious, biologically contained negative-sense, single stranded RNA virus, e.g., filovirus. In one embodiment, the method includes providing a host cell, e.g., a Vero cell, having a plurality of viral vectors which when expressed (stably or transiently) are effective to yield infectious, biologically contained negative-sense, single stranded RNA virus. In one embodiment, the plurality of vectors includes a vector for vRNA production comprising a promoter operably linked to a virus DNA which contains a deletion of sequences for a viral gene corresponding to Ebola virus VP30 which deletion is effective to prevent expression of a functional viral protein corresponding to Ebola virus VP30, linked to a transcription termination sequence. In another embodiment, the plurality of vectors includes a vector for vRNA production comprising a promoter operably linked to a viral DNA which contains a deletion of sequences for a viral gene corresponding to an Ebola virus L gene, which deletion is effective to prevent expression of a functional viral protein corresponding to Ebola virus L protein, linked to a transcription termination sequence. In another embodiment, the plurality of vectors includes a vector for vRNA production comprising a promoter operably linked to a viral DNA which contains a deletion of sequences for a viral gene corresponding to an Ebola virus VP30 gene and a viral gene

corresponding to an Ebola virus L gene, which deletions are effective to prevent expression of functional viral proteins corresponding to Ebola virus VP30 and L proteins, linked to a transcription termination sequence. The host cell also includes a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding a viral polymerase, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding viral nucleoprotein, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding one or more other viral proteins which along with the viral polymerase and nucleoprotein, are viral proteins needed for viral replication, and a vector comprising a promoter operably linked to a DNA encoding a RNA polymerase that is heterologous to the host cell. The heterologous RNA polymerase is selected to promote transcription of the viral DNA which contains the deletion. In one embodiment, the vector for vRNA includes a T7 polymerase promoter and a ribozyme sequence capable of cleaving a transcript to yield a vRNA-like 3' end. Then infectious, biologically contained virus is isolated from the cell. In one embodiment, the host cell is transiently transfected with the plurality of vectors and virus collected within 1, 2, 3, and up to 7 days post-transfection. hi one embodiment, the host cell is one that is approved for vaccine production, hi one embodiment, additional heterologous sequences are included in the vRNA vector and then along with the vectors for mRNA vectors subsequently introduced to the host cell, and/or are introduced to the host cell via a mRNA vector. In one embodiment, the additional heterologous sequences are for an immunogenic polypeptide or peptide of a pathogen, a tumor antigen, a therapeutic protein, or a reporter or selectable marker.

Further provided is a method to passage an infectious, biologically contained negative-sense, single stranded RNA virus of the invention, e.g., an infectious, biologically contained filovirus. The method includes providing a culture of mammalian cells that express (transiently or stably) a recombinant viral protein that is necessary in trans for viral replication. The mammalian cells are infected with the infectious, biologically contained virus of the invention so as to replicate and amplify the virus, e.g., to titers of at least 10 ⁶ FFU/mL, e.g., 10 ⁷ or greater FFU/niL. In one embodiment, the host cell is one that is approved for vaccine production. In one embodiment, the host cell expresses sequences

for filovirus genes corresponding to Ebola virus VP30, Ebola virus L, Ebola virus VP35, and/or Ebola virus NP genes, to provide the corresponding viral proteins, and is transfected with a filovirus genome having a deletion in at least one viral sequence corresponding to that for a nonstructural or nonglycosylated filovirus protein that is essential in trans for viral replication but which viral genome has sequences for filovirus genes corresponding to those for Ebola virus VP24 and VP40 proteins, and optionally for a homologous, e.g., Ebola virus, glycoprotein, or heterologous, e.g., chimeric, glycoprotein. Also provided is isolated infectious, biologically contained negative-sense, single stranded RNA virus prepared by the methods of the invention.

The invention also provides a method to immunize a mammal against negative-sense, single stranded RNA viruses. The method includes administering to the mammal an effective amount of a vaccine having the recombinant infectious, biologically contained virus of the invention. The recombinant infectious, biologically contained filovirus of the invention may also be employed to treat a mammal having been or suspected of being exposed to filovirus. The method includes administering an effective amount of the recombinant infectious, biologically contained virus of the invention to the mammal. For example, a human in contact with filovirus infected individuals or inadvertently exposed to filovirus, e.g., in a laboratory, may be administered the recombinant infectious, biologically contained virus of the invention in an amount effective to inhibit or substantially eliminate filovirus replication in the human.

The invention further provides screening methods that employ the recombinant infectious, biologically contained virus of the invention, hi one embodiment, the methods include those that identify one or more agents that inhibit virus infection or replication. The methods include contacting the recombinant infectious, biologically contained virus of the invention, a host cell, e.g., a helper cell, and one or more agents. Then it is determined whether the one or more agents inhibit viral replication or infection. Further provided is a method to identify one or more agents that inhibit virus infection or replication, which includes contacting a host cell infected with virus of the invention, or a lysate thereof, and one or more agents. Then it is determined whether the one or more agents inhibit viral replication or infection.

Brief Description of the Figures

Figure 1. Schematic diagram of EbolaδVP30 constructs. (Top row) Schematic diagram of the Ebolavirus genome flanked by the leader sequence (1) and the trailer sequence (t) in positive-sense orientation. Two unique restriction sites for SaIi and Sad (positions 6180 and 10942 of the viral antigenome, respectively) allowed the subcloning of a fragment that spans the VP30 gene. The subgenomic fragment was then used to replace the VP30 gene with genes encoding neomycin (neo) or enhanced green fluorescence protein (eGFP), respectively. Using the unique restriction sites, the altered subgenomic fragments were cloned back into the full-length Ebolavirus cDNA construct.

Figure 2. Characterization of EbolaδVP30-neo virus. (A) Expression of Ebolavirus antigens by infected VeroVP30 cells. Confluent VeroVP30 cells (left panel) or wild-type Vero cells (right panel) were infected with EbolaδVP30-neo for 60 minutes, washed, and overlaid with propagation medium with 1.5% methyl cellulose. Seven days later, cells were fixed with 10% buffered formaldehyde and an immunostaining assay with an antibody to Ebolavirus VP40 protein was performed. The formation of plaques in the VeroVP30 cell monolayer (left panel), but not in monolayers of wild-type Vero cells (right panel), illustrates thatEbolaδVP30-neo virus is biologically contained. (B) Detection of EbolaδVP30-neo viral proteins. Supernatants derived from infected VeroVP30 (labeled '+') or wild-type Vero (labeled '-') cells were collected 5 days after infection and partially purified over 20% sucrose. Protein pellets were suspended in PBS and separated on polyacrylamide gels, transferred to membranes and probed with specific antibodies to Ebolavirus proteins.

Figure 3. Replication kinetics of wild-type Ebolavirus and EbolaδVP30- neo virus. VeroVP30 cells (top panels) and wild-type Vero cells (bottom panels) were infected with Ebolavirus or EbolaδVP30-neo at a high m.o.i. of 1.0 (left panels) or a low m.o.i. of 0.01 (right panels). Supernatants were harvested every 24 hours postinfection for 6 days. Viral titers of the respective viruses were determined by infecting confluent VeroVP30 cells or wild-type Vero cells with tenfold dilutions of the supernatants and subsequent immunostaining. Virus titers for EbolaδVP30-neo virus (solid squares) and wild-type Ebolavirus (open

circles) were comparable in VeroVP30 cells (top panels). In wild-type Vero cells (bottom panels), no replication was detected for EbolaδVP30-neo virus (solid squares).

Figure 4. Morphology of Ebolaviruses budding from infected cells. Vero cells infected with wild-type Ebolavirus (left panels) and VeroVP30 cells infected with EbolaδVP30-neo virus (right panels) were processed for TEM 3 days postinfection. The pictures show virus budding from infected cells. No significant differences in morphology or budding efficiencies were observed for wild-type Ebolavirus and EbolaδVP30-neo virus. Top panel, 6,00Ox magnification; bottom panel, 20,00Ox magnification of boxed area from top panel.

Figure 5. Ebola δVP30 virus generates an antibody response against the Ebola virus glycoprotein, GP. (a) Flow chart of vaccination of 4-week-old Balb/c mice with EbolaδVP30 virus to determine the antibody titer to Ebola GP. Mice (n=4) were vaccinated three times with 10 ⁷ FFU of Ebola δVP30 at three- week intervals; control mice (n=4) were simultaneously mock-vaccinated. Serum samples were collected two weeks after each vaccination, (b) The amounts of IgG against purified Ebola virus GP in the samples was determined by ELISA. Results are expressed as the mean absorbance at 405 nm (+/- standard deviations) of samples diluted to 1 :100.

Figure 6. Cellular immune response in Ebola δVP30- vaccinated mice. Mice (n=4) were vaccinated with EbolaδVP30; control mice (n=2) were simultaneously mock- vaccinated. Splenocytes were collected 8 days after the second vaccination and stimulated with an NP peptide. Cells were stained for the cell surface antigen CD8 ⁺ and for intracellular IFNγ. The number of cytokine-producing CD8 ⁺ T cells was determined by using a FACSCalibur flow cytometer (BD Biosciences).

Figure 7. Flow chart of the vaccination schedule to determine the protective efficacy of the EbolaδVP30 virus. Four-week-old Balb/c mice were vaccinated with EbolaδVP30 virus, hi group 1, mice (n=14) were vaccinated with nonpurified EbolaδVP30 virus directly from cell culture supernatant, while control mice (n=8) were mock- vaccinated. In group 2, mice (n=15) were vaccinated with purified EbolaδVP30 virus, while control mice (n=10) were

mock-vaccinated. All mice were challenged with a 1000 MLD ₅ O of mouse- adapted Ebola virus.

Figure 8. Body weight changes (a) and Kaplan-Meier survival curve (b) of mice vaccinated with EbolaδVP30 compared to control mice. Mice from group 1 were vaccinated three times with non-purified EbolaδVP30 virus while mice from group 2 were vaccinated twice with purified EbolaδVP30 virus. Mice from the vaccinated groups and control groups were challenged with a 1000 MLD _5O of mouse-adapted Ebola virus.

Figure 9. Virus titers in the serum of mice following lethal challenge. Vaccinated (n=3) and control (n=3) mice from groups 1 and 2 were euthanized on day 4 post-challenge. Virus titers from the serum were determined by the plaque assay. ND, not detectable.

Figure 10. Representative fϊlovirus sequences (Accession numbers NC006432, NC004161, AY769362, AY142960, AF522874, AF499101, Ll 1365, NCOOl 608, DQ447652, DQ447649, AB050936, NC002549,

NC001608, AF086833 and AF272001, the disclosures of which are incorporated by reference herein; SEQ ID Nos.1-15 and 18-40).

Figure 11. Flow chart of the vaccination schedule to determine the protective efficacy of the EbolaδVP30 virus in guinea pigs. Guinea pigs were vaccinated with 1 x 10 ⁷ FFU EbolaδVP30 virus or mock-vaccinated at day zero and day 21. Forty-two days after the second vaccination or mock-vaccination, guinea pigs were challenged with 1 ,000 MLD ₅₀ of Ebola virus.

Figure 12. Graph of percent survival versus day after challenge of EbolaδVP30 vaccinated or mock-vaccinated guinea pigs with 1000 MLD ₅₀ of Ebola virus.

Figure 13. Body weight changes in guinea pigs vaccinated with EbolaδVP30 and challenged with Ebola virus compared to control guinea pigs.

Figure 14. Schematic diagram of Ebola virus wild-type, EbolaδL-eGFP virus and EbolaδVP30-neo δL-eGFP virus genomes flanked by the leader sequence (1) and the trailer sequence (T) in positive-sense orientation.

Figure 15. Rescue of EbolaδL-eGFP in 293-VP30/L cell line. A) 293- VP30/L cells were transfected with a plasmid expressing the Ebola genome lacking the L gene (pTM EbolaδL) along with protein expression plasmids for

NP, VP35, and T7 polymerase. B) As a control, 293-VP30/L cells were transfected with pTM EbolaδL and T7 polymerase without NP and VP35. Figure 16. Protective efficacy of EbolaδVP30 virus in guinea pigs. Survival curves of guinea pigs vaccinated twice with EbolaδVP30 virus or vaccinated with PBS (n=6 per group). Forty-two days after the last vaccination, guinea pigs were challenged with 1,000 LD ₅₀ of guinea pig adapted-Ebola virus. Animals were observed for 16 days after challenge.

Detailed Description of the Invention Definitions

A "vector" or "construct" (sometimes referred to as gene delivery or gene transfer "vehicle") refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. The polynucleotide to be delivered may comprise a coding sequence of interest for gene therapy. Vectors include, for example, viral vectors (such as adenoviruses, adeno-associated viruses (AAV), lentiviruses, herpesvirus and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell. Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide. Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. A large variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, the vector can either be stably replicated by

the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell's nucleus or cytoplasm. A "recombinant viral vector" refers to a viral vector comprising one or more heterologous genes or sequences. Since many viral vectors exhibit size constraints associated with packaging, the heterologous genes or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-defective (biologically contained), requiring the deleted function(s) to be provided in trans during viral replication and encapsidation (by using, e.g., a helper virus or a packaging cell line carrying genes necessary for replication and/or encapsidation). Modified viral vectors in which a polynucleotide to be delivered is carried on the outside of the viral particle have also been described.

"Gene delivery," "gene transfer," and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art.

By "transgene" is meant any piece of a nucleic acid molecule (for example, DNA) which is inserted by artifice into a cell either transiently or permanently, and becomes part of the organism if integrated into the genome or maintained extrachromosomally. Such a transgene may include at least a portion of an open reading frame of a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent at least a portion of an open reading frame of a gene homologous to an endogenous gene of the

organism, which portion optionally encodes a polypeptide with substantially the same activity as the corresponding full-length polypeptide or at least one activity of the corresponding full-length polypeptide.

By "transgenic cell" is meant a cell containing a transgene. For example, a cell stably or transiently transformed with a vector containing an expression cassette is a transgenic cell that can be used to produce a population of cells having altered phenotypic characteristics. A "recombinant cell" is one which has been genetically modified, e.g., by insertion, deletion or replacement of sequences in a nonrecombinant cell by genetic engineering. The term "wild-type" or "native" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The term "transduction" denotes the delivery of a polynucleotide to a recipient cell either in vivo or in vitro, via a viral vector and preferably via a replication-defective viral vector.

The term "heterologous" as it relates to nucleic acid sequences such as gene sequences encoding a protein and control sequences, denotes sequences that are not normally joined together, and/or are not normally associated with a particular cell, e.g., are from different sources (for instance, sequences from a virus are heterologous to sequences in the genome of an uninfected cell). Thus, a "heterologous" region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature, i.e., a heterologous promoter. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic

sequences having codons different from the native gene). Similarly, a cell transformed with a construct which is not normally present in the cell would be considered heterologous for purposes of this invention.

By "DNA" is meant a polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in double-stranded or single-stranded form found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having the sequence complementary to the mRNA). The term captures molecules that include the four bases adenine, guanine, thymine, or cytosine, as well as molecules that include base analogues which are known in the art.

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.

DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide or polynucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or

5' of the "downstream" or 3 ¹ elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements that direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

A "gene," "polynucleotide," "coding region," "sequence," "segment, " "fragment" or "transgene" which "encodes" a particular protein, is a nucleic acid molecule which is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence.

The term "control elements" refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, splice junctions, and the like, which collectively provide for the replication, transcription, post-transcriptional processing and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence.

By "enhancer" is meant a nucleic acid sequence that, when positioned proximate to a promoter, confers increased transcription activity relative to the transcription activity resulting from the promoter in the absence of the enhancer domain. By "operably linked" with reference to nucleic acid molecules is meant that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and an enhancer element) are connected in such a way as to permit transcription of the nucleic acid molecule. "Operably linked" with reference to peptide and/or polypeptide molecules is meant that two or more peptide and/or polypeptide molecules are connected in such a way as to yield a single polypeptide chain, i.e., a fusion polypeptide, having at least one property of each peptide and/or polypeptide component of the fusion. The fusion polypeptide is preferably chimeric, i.e., composed of heterologous molecules. "Homology" refers to the percent of identity between two polynucleotides or two polypeptides. The correspondence between one sequence and to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single strand-specific nuclease(s), and size determination of the digested fragments. Two DNA, or two polypeptide, sequences are "substantially homologous" to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides, or amino acids, respectively match over a defined length of the molecules, as determined using the methods above.

By "mammal" is meant any member of the class Mammalia including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, rabbits and guinea pigs, and the like.

By "derived from" is meant that a nucleic acid molecule was either made or designed from a parent nucleic acid molecule, the derivative retaining

substantially the same functional features of the parent nucleic acid molecule, e.g., encoding a gene product with substantially the same activity as the gene product encoded by the parent nucleic acid molecule from which it was made or designed. By "expression construct" or "expression cassette" is meant a nucleic acid molecule that is capable of directing transcription. An expression construct includes, at the least, a promoter. Additional elements, such as an enhancer, and/or a transcription termination signal, may also be included.

The term "exogenous," when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell. By way of a non-limiting example, an exogenous nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.

The term "isolated" when used in relation to a nucleic acid, peptide, polypeptide or virus refers to a nucleic acid sequence, peptide, polypeptide or virus that is identified and separated from at least one contaminant nucleic acid, polypeptide or other biological component with which it is ordinarily associated in its natural source, e.g., so that it is not associated with in vivo substances, or is substantially purified from in vitro substances. Isolated nucleic acid, peptide, polypeptide or virus is present in a form or setting that is different from that in which it is found in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. The isolated nucleic acid molecule may be present in single-stranded or double-stranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the molecule will contain at a minimum the sense or coding strand (i.e., the molecule may single-stranded), but may contain both the sense and anti-sense strands (i.e., the molecule may be double- stranded).

As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence, molecule or segment" refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from a source, that may be subsequently chemically altered in vitro, and includes, but is not limited to, a sequence that is naturally occurring, is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in the native genome. An example of DNA "derived" from a source, would be a DNA sequence that is identified as a useful fragment, and which is then chemically synthesized in essentially pure form. An example of such DNA "isolated" from a source would be a useful DNA sequence that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.

The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule that is expressed from a recombinant DNA molecule.

The term "peptide", "polypeptide" and protein" are used interchangeably herein unless otherwise distinguished.

The term "sequence homology" means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred. When using oligonucleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and more preferably not less than 19 matches out of 20 possible base pair matches (95%).

The term "selectively hybridize" means to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments of the invention selectively hybridize to nucleic acid strands under hybridization and wash

conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the invention and a nucleic acid sequence of interest is at least 65%, and more typically with preferably increasing homologies of at least about 70%, about 90%, about 95%, about 98%, and 100%.

Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.

The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (e.g., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence that encodes a polypeptide or its complement, or that a polypeptide sequence is identical in sequence or function to a reference polypeptide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".

The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene

sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.

A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by using local homology algorithms or by a search for similarity method, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA Genetics Software Package or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denote a characteristic of a

polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 80% sequence identity, preferably at least about 90% sequence identity, more preferably at least about 95%percent sequence identity, and most preferably at least about 99% sequence identity.

A "protective immune response" and "prophylactic immune response" are used interchangeably to refer to an immune response which targets an immunogen to which the individual has not yet been exposed or targets a protein associated with a disease in an individual who does not have the disease, such as a tumor associated protein in a patient who does not have a tumor.

A "therapeutic immune response" refers to an immune response which targets an immunogen to which the individual has been exposed or a protein associated with a disease in an individual who has the disease.

The term "prophylactically effective amount" is meant to refer to the amount necessary to, in the case of infectious agents, prevent an individual from developing an infection, and in the case of diseases, prevent an individual from developing a disease.

The term "therapeutically effective amount" is meant to refer to the amount necessary to, in the case of infectious agents, reduce the level of infection in an infected individual in order to reduce symptoms or eliminate the infection, and in the case of diseases, to reduce symptoms or cure the individual.

"Inducing an immune response against an immunogen" is meant to refer to induction of an immune response in a naive individual and induction of an

immune response in an individual previously exposed to an immunogen wherein the immune response against the immunogen is enhanced.

As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, about 90%, about 95%, and about 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. "Transfected," "transformed" or "transgenic" is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells of the present invention are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or infection with a recombinant viral vector.

Exemplary Viruses and Methods of the Invention

The invention provides isolated vectors, e.g., plasmids, which encode proteins of negative-sense, single stranded RNA viruses and/or express vRNA from recombinant nucleic acid corresponding to sequences for mutant negative- sense, single stranded RNA viruses. When introduced into a cell, a combination of these vectors is capable of yielding recombinant infectious, biologically contained virus. Thus, the invention includes host cells that produce recombinant infectious, biologically contained virus of the invention. In one embodiment, the invention provides isolated vectors, e.g., plasmids, which encode filovirus proteins and/or express mutant filovirus vRNA which, when introduced into a cell, are capable of yielding recombinant infectious, biologically contained filovirus. The invention includes host cells that transiently or stably produce recombinant infectious, biologically contained

filovirus, including helper cells, and isolated recombinant filovirus prepared by the methods disclosed herein.

The vectors of the invention include those for mRNA production and vRNA production. In one embodiment, the vectors include filovirus DNA, for example, vectors for mRNA production with sequences corresponding to one or more open reading frames encoding filovirus proteins, or vectors for vRNA production that include a deletion of the full-length genomic sequence, which deletion includes internal filovirus sequences corresponding to at least a portion of one open reading frame. The RNA produced from the vRNA vector is capable of being packaged into virions in the presence of filovirus proteins but as part of the resulting virion, is not capable of being replicated and so does not result in virus production when that virion is introduced to a cell that otherwise supports filovirus replication and which cell does not express at least one filovirus protein in trans, e.g., a cell that is not a filovirus helper cell. For example, Ebolaviruses possess a negative-sense, nonsegmented RNA genome, approximately 19 kilobases in length that encodes seven structural proteins and at least one non-structural protein (Sanchez et al., 2007). NP, viral protein (VP)35, VP30, and L, the RNA-dependent RNA polymerase, are components of the nucleocapsid involved in viral replication and transcription (Muhlberger et al., 1999). VP40 is the matrix protein and is involved in viral budding (Harty et al., 2000; Panchal et al., 2003). VP24 is involved in the formation of nucleocapsids composed of NP, VP35 and viral RNA (Huang et al., 2002). The only viral surface glycoprotein, GP, plays a role in viral attachment and entry (Chan et al., 2001; Manicassamy et al., 2005; Shimojima et al., 2006; Chandran et al., 2005). Candidate sequences for deletion/mutation and optional replacement with heterologous sequences include but are not limited to Ebola virus VP30 sequences or corresponding sequences in other negative-sense, single stranded RNA viruses, e.g., sequences for nonstructural, nonpolymerase and/or nonglycosylated viral proteins. Although deletions in other Ebola virus sequences, i.e., in GP and VP40, were prepared, only deletions in VP30 sequences resulted in virus that could be recovered. However, deletions in sequences that do not correspond to VP30 sequences in other negative-sense, single stranded RNA viruses may yield infectious, biologically contained virus that is useful in vaccines or in drug screening.

The vectors may include gene(s) or portions thereof other than those of a negative-sense, single stranded RNA virus such as a filovirus (heterologous sequences), which genes or portions thereof are intended to be expressed in a host cell, either as a protein or incorporated into vRNA. Thus, a vector of the invention may include in addition to viral sequences, for instance, filovirus sequences, a gene or open reading frame of interest, e.g., a heterologous gene for an immunogenic peptide or protein useful as a vaccine or a therapeutic protein.

If more than one vector is employed, the vectors may be physically linked or each vector may be present on an individual plasmid or other, e.g., linear, nucleic acid delivery vehicle. The vectors or plasmids may be introduced to any host cell, e.g., a eukaryotic cell such as a mammalian cell, that supports viral replication. Host cells useful to prepare virus of the invention include but are not limited to insect, avian or mammalian host cells such as canine, feline, equine, bovine, ovine, or primate cells including simian or human cells. In one embodiment, the host cell is one that is approved for vaccine production.

The viruses produced by methods described herein are useful in viral mutagenesis studies, drug screening and in the production of vaccines (e.g., for AIDS, influenza, hepatitis B, hepatitis C, rhinovirus, filoviruses, malaria, herpes, and foot and mouth disease) and gene therapy vectors (e.g., for cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamylase deficiency and central nervous system tumors). In particular, infectious, biologically contained filovirus of the invention which induces strong humoral and cellular immunity may be employed as a vaccine vector, as they are unlikely to give rise to infectious recombinant virus. Thus, a virus for use in medical therapy (e.g., for a vaccine or gene therapy) is provided. For example, the invention provides a method to immunize an animal against a pathogen, e.g., a bacteria, virus such as Ebola virus, or parasite, or a malignant tumor. The method comprises administering to the animal an effective amount of at least one isolated virus of the invention which encodes and expresses, or comprises nucleic acid for an immunogenic peptide or protein of a pathogen or tumor, optionally in combination with an adjuvant, effective to immunize the animal.

To prepare expression cassettes for transformation herein, the recombinant DNA sequence or segment may be circular or linear, double-

stranded or single-stranded. A DNA sequence which encodes an RNA sequence that is substantially complementary to a mRNA sequence encoding a gene product of interest is typically a "sense" DNA sequence cloned into a cassette in the opposite orientation (i.e., 3N to 5N rather than 5N to 3N). Generally, the DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the DNA in a cell. As used herein, "chimeric" means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the "native" or wild-type of the species.

Aside from DNA sequences that serve as transcription units, or portions thereof, a portion of the DNA may be untranscribed, serving a regulatory or a structural function. For example, the DNA may itself comprise a promoter that is active in eukaryotic cells, e.g., mammalian cells, or in certain cell types, or may utilize a promoter already present in the genome that is the transformation target of the lymphotropic virus. Such promoters include the CMV promoter, as well as the SV40 late promoter and retroviral LTRs (long terminal repeat elements), e.g., the MMTV, RSV, MLV or HIV LTR, although many other promoter elements well known to the art may be employed in the practice of the invention.

Other elements functional in the host cells, such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the recombinant DNA. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.

The recombinant DNA to be introduced into the cells may contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are

well known in the art and include, for example, antibiotic and herbicide- resistance genes, such as neo, hpt, dhfr, bar, aroA, puro, hyg, dapA and the like. See also, the genes listed on Table 1 of Lundquist et al. (U.S. Patent No. 5,848,956). Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Exemplary reporter genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the beta- glucuronidase gene (gus) of the uidA locus of E. coli, the green, red, or blue fluorescent protein gene, and the luciferase gene. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

The general methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful herein. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2002) provides suitable methods of construction.

The recombinant DNA can be readily introduced into the host cells, e.g., mammalian, yeast or insect cells, by transfection with an expression vector comprising the recombinant DNA by any procedure useful for the introduction into a particular cell, e.g., physical or biological methods, to yield a transformed (transgenic) cell having the recombinant DNA so that the DNA sequence of interest is expressed by the host cell. In one embodiment, at least one of the recombinant DNA which is introduced to a cell is maintained extrachromosomally. In one embodiment, at least one recombinant DNA is stably integrated into the host cell genome. Physical methods to introduce a recombinant DNA into a host cell include calcium-mediated methods, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods to introduce the DNA of interest into a host cell include the use of DNA and RNA viral vectors. Viral vectors, e.g., retroviral or lentiviral vectors, have become a widely

used method for inserting genes into eukaryotic, such as mammalian, e.g., human, cells. Other viral vectors useful to introduce genes into cells can be derived from poxviruses, e.g., vaccinia viruses, herpes viruses, adenoviruses, adeno-associated viruses, baculoviruses, and the like. To confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular gene product, e.g., by immunological means (ELISAs and Western blots) or by other molecular assays.

To detect and quantitate RNA produced from introduced recombinant DNA segments, RT-PCR may be employed. In this application of PCR, it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique demonstrates the presence of an RNA species and gives information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and only demonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the recombinant DNA segment in question, they do not provide information as to whether the recombinant DNA segment is being expressed. Expression may be evaluated by specifically identifying the peptide products of the introduced DNA sequences or evaluating the phenotypic changes brought about by the expression of the introduced DNA segment in the host cell.

The recombinant viruses described herein have modifications in genomic sequences relative to a corresponding wild-type viral genome, i.e., the genome of the recombinant virus has a modification which includes a deletion, and optionally an insertion, in a region corresponding to sequences for a viral protein that is associated with transcription, is nonstructural or nonglycosylated. The mutation in the viral genome is effective to inhibit or prevent production of at

least one functional viral protein from that genome when those sequences are present in a nontransgenic cell which supports viral replication. In one embodiment, the deletion includes from 1 up to thousands of nucleotides, e.g., 1%, 10%, 50%, 90% or more of sequences corresponding to the coding region for the viral protein. In one embodiment, the deleted sequences correspond to sequences with a substantial identity, e.g., at least 80% or more, e.g., 85%, 90% or 95% and up to 100% or any integer in between, nucleic acid sequence identity, to VP30 sequences.

In one embodiment, the viral genome in an infectious, replication- incompetent negative-sense, single-stranded RNA virus of the invention includes a deletion in sequences corresponding to those in a wild-type viral genome for a protein that is associated with transcription or is nonstructural or nonglycoslyated, and includes heterologous sequences that are nontoxic to host cells including cells in an organism to be immunized. In one embodiment, the heterologous sequence is a marker sequence, a selectable sequence or other sequence which is detectable or capable of detection, e.g., GFP or luciferase, or a selectable gene such as an antibiotic resistance gene, e.g., a hygromycin B resistance gene or neomycin phosphotransferase gene, which marker gene or selectable gene is not present in the host cell prior to introduction of the vector. Pharmaceutical Compositions

Pharmaceutical compositions of the present invention, suitable for inoculation, e.g., nasal, parenteral or oral administration, such as by intravenous, intramuscular, topical or subcutaneous routes, comprise one or more virus isolates, e.g., one or more recombinant infectious, biologically contained negative-sense, single stranded RNA virus isolates, optionally further comprising sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions can further comprise auxiliary agents or excipients, as known in the art. The composition of the invention is generally presented in the form of individual doses (unit doses). Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin

permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

When a composition of the present invention is used for administration to an individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. For vaccines, adjuvants, substances which can augment a specific immune response, can be used. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the organism being immunized. In one embodiment, the pharmaceutical composition is part of a controlled release system, e.g., one having a pump, or formed of polymeric materials (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger & Peppas, J. Macromol. Sci. Rev. Macromol. Chem.,

23:61 (1983); see also Levy et al., Science. 228:190 (1985); During et al., Ann. Neurol.. 25:351 (1989); Howard et al., J. Neurosurg.. 71:105 (1989)). Other controlled release systems are discussed in the review by Langer (Science. 249:1527 (1990)). The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of the virus, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt,

rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. These compositions can be formulated as a suppository. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the virus, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

The compositions may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent. For oral administration, the virus may be combined with one or more excipients and used in the form of ingestible capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such useful compositions is such that an effective dosage level will be obtained.

The compositions may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. Various other materials may be present. For instance, a syrup or elixir may contain the virus, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form, including sustained-release preparations or devices, should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. The composition also be administered

intravenously or intraperitoneally by infusion or injection. Solutions of the virus can be prepared in water or a suitable buffer, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of undesirable microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of undesirable microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride.

Sterile injectable solutions are prepared by incorporating the virus in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.

Useful liquid carriers include water, alcohols or glycols or water- alcohol/glycol blends, in which the present viruses can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Useful dosages of the viruses of the invention can be determined by comparing their in vitro activity and in vivo activity in animal models.

Pharmaceutical Purposes

The administration of the composition may be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the compositions of the invention which are vaccines are provided before any symptom or clinical sign of a pathogen infection becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. When provided prophylactically, the gene therapy compositions of the invention, are provided before any symptom or clinical sign of a disease becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or clinical signs associated with the disease.

When provided therapeutically, a viral vaccine is provided upon the detection of a symptom or clinical sign of actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. When provided therapeutically, a gene therapy composition is provided upon the detection of a symptom or clinical sign of the disease. The therapeutic administration of the compound(s) serves to attenuate a symptom or clinical sign of that disease.

Thus, a vaccine composition of the present invention may be provided either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection. Similarly, for gene therapy, the composition may be provided before any symptom or clinical sign of a disorder or disease is manifested or after one or more symptoms are detected.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. A composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular immune response against at least one strain of a virus.

The "protection" provided need not be absolute, i.e., the influenza infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of mammals.

Protection may be limited to mitigating the severity or rapidity of onset of symptoms or clinical signs of the virus infection. Pharmaceutical Administration

A composition of the present invention may confer resistance to one or more pathogens, e.g., one or more virus strains, by either passive immunization or active immunization. In active immunization, a live vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host's immune response to the administration protects against infection and/or disease. For passive immunization, the elicited antisera can be recovered and administered to a recipient suspected of having an infection caused by at least one virus strain.

The present invention thus includes methods for preventing or attenuating a disorder or disease, e.g., an infection by at least one strain of pathogen. As used herein, a vaccine is said to prevent or attenuate a disease if its administration results either in the total or partial attenuation (i.e., suppression) of a clinical sign or condition of the disease, or in the total or partial immunity of the individual to the disease.

At least one virus isolate of the present invention, may be administered by any means that achieve the intended purposes. For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be accomplished by bolus injection or by gradual perfusion over time.

A typical regimen for preventing, suppressing, or treating a viral related pathology, comprises administration of an effective amount of a vaccine composition as described herein, administered as a single treatment, or repeated as enhancing or booster dosages, for instance, over a period up to and including between one week and about 24 months, or any range or value therein. According to the present invention, an "effective amount" of a composition is one that is sufficient to achieve a desired effect. It is understood that the effective dosage may be dependent upon the species, age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted. The ranges of effective doses provided below are not intended to limit the invention and represent dose ranges.

The invention will be further described in the following nonlimiting examples.

Example 1 Methods and Materials

Cells and cell lines. Vero cells (green monkey kidney cells) were grown in Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), L-glutamine, vitamins, nonessential amino acid solution and antibiotics. The VeroVP30 cell line was established by cotransfecting Vero cells with pCAG-VP30 (for the expression of VP30) and pPur, a protein expression plasmid for the puromycin resistance gene (Clontech, Mountain View, CA), using the transfection reagent TransIT LT-I (Minis, Madison, WI). Two days after transfection, puromycin-resistant cells were selected with 5 μg/mL puromycin (Sigma, St. Louis, MO). Individual cell clones were screened for VP30 expression by flow cytometry with a polyclonal peptide antibody to VP30.

Human embryonic kidney 293 T cells were grown in high- glucose Dulbecco's modified Eagle medium containing 10% FCS, L-glutamine, and antibiotics. All cells were maintained at 37°C and 5% CO ₂ .

Flow cytometry. Cells were detached in phosphate-buffered saline (PBS) containing 0.02% EDTA and then washed once with cold PBS supplemented with 2% FCS and 0.1% sodium azide (wash buffer). Cells were incubated with a VP30 antibody on ice for 20 minutes. After washing in buffer, the cells were further incubated with a secondary antibody labeled with fluorescent isothiocyanate (Zymed Laboratories, Carlsbad, CA). They were then washed with buffer and analyzed byFACSCalibur with Cell Quest software (Becton Dickinson, Franklin Lakes, NJ).

Generation of EbolaδVP30 viruses. The plasmid pTM-T7G-Ebo-Rib, containing the full-length Ebolavirus cDNA flanked by T7 RNA polymerase promoter and ribozyme sequences, is described in Newmann et al. (2002). First, a fragment encompassing nucleotides 6180 to 10942 (numbers refers to the positive-sense antigenome) was subcloned into a kanamycin-resistant cloning vector. Next, the VP30 ORF was replaced with those encoding neo or eGFP, respectively, by a series of overlapping PCR amplification steps using Pfu Turbo (Stratagene, La Jolla, CA). The altered subgenomic fragments were transferred

back into the full-length Ebolavirus cDNA plasmid using two unique restriction sites, Sail and Sad (Figure 1). The resultant plasmids, designated pTM- EbolaδVP30-neo or -eGFP, were sequenced to verify the replacement of the VP30 ORF and the lack of any unwanted mutations. To artificially generate Ebolavirus, 5 x 10 ⁵ 293T cells were transfected with 1.0 μg pTM-EbolaδVP30, 2.0 μg pCAG-L, 1.0 μg ρCAG-NP, 0.5 μg pCAG-VP35, 0.5 μg pCAG-VP30, and 1.0 μg pCAG-T7 pol, using TransIT LTl (Mirus, Madison, WI) in BSL-4 containment (Neumann et al., 2002). Five days after transfection, the supernatant was harvested, cellular debris removed by low speed centrifugation, and the virus amplified in VeroVP30 cells at 37°C and 5% CO ₂ with propagation medium containing 2% FCS in MEM supplemented with L-glutamine, vitamins, nonessential amino acid solution and antibiotics without puromycin.

Plaque assay and immunostaining assay. To determine the titers of wild- type Ebolavirus or EbolaδVP30 viruses, tenfold dilutions of the viruses were absorbed to confluent VeroVP30 or wild-type Vero cells for 1 hour at 37°C, after which any unbound virus was removed by washing cells with propagation medium. The cells were then overlaid with propagation medium containing 1.5% methyl cellulose (Sigma). Seven days after infection, cells were fixed with 10% buffered formaldehyde, taken out of BSL-4, permeabilized with 0.25% Triton X-100 in PBS for 10 minutes, and blocked with 4% goat serum and 1% bovine serum albumin (BSA) in PBS for 60 minutes. Cells were then incubated for 60 minutes with a 1 : 1000 dilution of a mouse anti-VP40 monoclonal antibody, washed with PBS, and incubated for 60 minutes with a 1 :1000 dilution of an antimouse IgG-peroxidase-conjugated secondary antibody (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). After washing with PBS, cells were incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma) in PBS. The reaction was stopped by rinsing cells with water.

Western blotting. Partially purified virus resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 % Triton X-100, and 0.1% SDS) containing protease inhibitors (complete protease inhibitor cocktails [Roche]) was incubated at 100 ⁰ C for 5 minutes, taken out of BSL-4, and separated on 4- 20% polyacrylamide gels. Resolved proteins were transferred to Western polyvinylidine difluoride membranes (Schleicher & Schuell, Sanford, ME) and

blocked overnight at 4°C with 5% skim milk in PBST (0.05% Tween 20 [Sigma] in PBS). Blots were incubated with primary antibodies (a mouse anti-NP antibody, a rabbit anti-VP35 antibody, a rabbit anti-VP40 antibody, a mouse anti-GP antibody, a rabbit anti-VP30 antibody, or a mouse anti-VP24 antibody) for 60 minutes at room temperature, washed three times with PBST, incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (Zymed) for 60 minutes, and finally washed three times with PBST. Blots were then incubated in Lumi-Light Western blotting substrate (Roche, Indianapolis, IN) and exposed to X-ray film (Kodak, Rochester, NY). RNA isolation and RT-PCR. Cell culture supernatant from virus- infected VeroVP30 cells was inactivated with guanidinium isothiocyanate buffer and taken out of BSL-4. Viral RNA was isolated with the RNeasy Mini kit (Qiagen, Valencia, CA). RT-PCR was carried out with the RobusT One-Step RT-PCR kit (Finnzyme, Espoo, Finland), using 1 μg of isolated RNA and Ebolavirus-spQcific primers. The resultant PCR products were cloned into pT7Blue (Novagen, San Diego, CA) and sequenced.

Transmission electron microscopy. Ultrathin-section electronmicroscopy was performed as described in Noda et al. (2002). Briefly, at 36 hours postinfection, VeroVP30 cells infected with EbolaδVP30-neo virus were fixed and inactivated with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, taken out of BSL-4 and postfixed with 2% osmium tetroxide in the same buffer. Cells were then dehydrated with a series of ethanol gradients followed by propylene oxide, before being embedded in Epon 812 Resin mixture (TAAB Laboratories Equipment Ltd., Berkshire, UK). Thin sections were stained with 2% uranyl acetate and Raynold's lead, and examined under a HITACHI H-7500 electron microscope at 80 kV.

Selection of escape mutants. EbolaδVP30-eGFP was diluted tenfold (10 ^{" 1} to 10 ^"6 ) and incubated with the indicated mAbs at a concentration of 250 to 500 μg of mAb/niL at 37 ⁰ C for 60 minutes. The virus/mAb mixtures were inoculated onto VeroVP30 cells for 60 minutes. Viruses were amplified for 5 days in the presence of antibodies. Then, viruses that grew in the presence of mAbs (as determined by GFP expression) were harvested at the highest virus-positive dilution and passaged for a total of 3-6 times in the presence of antibodies. Viral

RNA was isolated, RT-PCR amplified, and the GP sequence determined by sequence analysis.

Results

Generation and passage of EbolaδVP30-neo virus. Previously a full- length cDNA clone of the Zaire ebolavirus-Mayinga. was generated (Newmann et al., 2002). Using a subgenomic fragment that encompasses nucleotides 6180 to 10942 of the viral genome (numbers refers to the positive-sense antigenome), the ORF for VP30 was replaced with that of neomycin (neo), using a series of overlapping PCR amplification steps. After confirmation of the authenticity of the PCR fragments by sequence analysis, the altered subgenomic fragment was inserted into the full-length Ebolαvirus cDNA construct via unique Sail and Sad restriction sites (Figure 1), resulting in an Ebolavirus cDNA genome deficient in the VP30 ORF. The artificial generation of Ebolavirus from plasmids is afforded by flanking this viral cDNA with T7 RNA polymerase promoter and hepatitis delta virus ribozyme sequences (Neumann et al., 2002).

To amplify VP30-deficient Ebolavirus es, a stable Vero E6 cell line (designated VeroVP30) was established by cotransfecting Vero cells with two protein expression plasmids encoding VP30 (pCAG-VP30) and puromycin (pPur, Clontech), and selecting cell clones resistant to 5.0 μg/mL of puromycin. VP30 expression in individual clones was determined by flow cytometry with antibodies to VP30. The clone with the highest percentage of VP30-expressing cells (> 90% as measured by flow cytometry, data not shown) was used in further studies to amplify EbolaδVP30 viruses.

EbolaδVP30-neo virus was rescued under BSL-4 conditions as described for wild-type Ebolavirus (Neumann et al., 2002). All work involving infectious EboδVP30 viruses and all steps prior to inactivation of biological material were performed under BSL-4 conditions at the National Microbiology Laboratory of the Public Health Agency of Canada.

Briefly, human embryonic kidney (293T) cells were transfected with a plasmid for the transcription of the VP30-deficient Ebolavirus RNA, with plasmids for the expression of the Ebolavirus NP, VP30, VP35, and L proteins, and with a plasmid for the expression of T7 RNA polymerase. Five days after transfection, VeroVP30 cells were incubated with undiluted supernatant derived from plasmid-transfected cells. Seven days later, the supernatant was harvested,

diluted tenfold, and used to infect fresh VeroVP30 cells for the next passage. A total of seven passages were carried out, using the highest dilution of the inoculum that still produced replicating viruses for each passage. The presence of replicating virus was assessed by cytopathic effects (CPE) and immunostaining of infected VeroVP30 cells with an antibody to VP40 (Figure 2A, left panel). As a control, we also incubated the supernatants from each passage with wild-type Vero cells. As expected, CPE and viral antigens were undetectable in wild-type Vero cells (Figure 2A, right panel), demonstrating that replicating EbolaδVP30-neo virus was confined to VeroVP30 cells. Although the manifestation of a CPE in infected VeroVP30 cells suggested the formation of infectious (but biologically contained) Ebolaviruses, further evidence was sought for the presence of virions in cell culture supernatant derived from infected VeroVP30 cells. Briefly, 5 days after VeroVP30 cells were infected with EbolaδVP30-neo virus, supernatant was collected and partially purified over 20% sucrose. The pellet was suspended in PBS and separated on a 4-20% polyacrylamide gel. Western blot analyses were carried out with antibodies specific to the respective Ebolavirus protein. All viral proteins (with the exception of L, for which no antibody was available) were detected (Figure 2B, '+' lanes). Note that VP30 protein in virions originates from VeroVP30 cells while the remaining proteins are encoded by EbolaδVP30- neo virus. By contrast, no viral proteins were detected in a control sample derived from wild- type Vero cells infected with EbolaδVP30-neo virus (Figure 2B, '-' lanes).

Genetic stability of EbolaδVP30-neo virus. A major concern with the use of VP30-defϊcient Ebolaviruses is the potential recombination with VP30 sequences integrated into the genome of the VeroVP30 helper cell line. Thus, to assess the genomic stability of EbolaδVP30-neo virus, three independent passage experiments were performed (seven passages each). While EbolaδVP30-neo virus replicated in VeroVP30 cells, viral replication was not observed in wild-type Vero cells. Total viral RNA was isolated from the cell culture supernatant of infected VeroVP30 cells after the seventh passage. A viral genomic fragment spanning the neo gene was amplified by RT-PCR, cloned and sequenced. A total of 20 clones were sequenced, and the sequences were identical to that of the EbolaδVP30 cDNA construct used for virus

generation. Hence, there was no evidence of recombination in any of three independent passage experiments, attesting to the genomic stability of the EbolaδVP30-neo viral genome.

To further demonstrate the biosafety of EbolaδVP30-neo virus, EbolaδVP30-neo virus was collected after seven consecutive passages in VeroVP30 cells and this virus used for three consecutive "blind" passages in wild-type Vero cells. Briefly, Vero cells were infected at a multiplicity of infection (m.o.i.) of 5 with EbolaδVP30-neo virus (passage 7). Six days later, supernatant was used for the next "blind" passage as well as for Western blot analysis. No viral NP protein was detected after any of the "blind" passages (data not shown). After three consecutive "blind" passages, plaque assays and immunostaining were carried out in wild-type Vero cells to confirm the absence of replicating Ebolavirus. As expected, replicating virus was not detected (data not shown). Collectively, these data further attest to the biosafety of the EbolaδVP30 system.

Growth kinetics of EbolaδVP30-neo virus. One of the major concerns raised by providing viral proteins in trans is that their amounts, expression kinetics or both may not match those found in cells infected with wild-type virus, leading to reduced virus titers and/or aberrant virion morphology. To address this potential pitfall, the growth kinetics of EbolaδVP30-neo virus (Figure 3, solid squares) were compared with that of wild-type Ebolavirus (Figure 3, open circles). VeroVP30 cells (Figure 3, top panels) or wild-type Vero cells (Figure 3, bottom panels) were infected at a high m.o.i. of 1.0 or a low m.o.i. of 0.01 and supernatant was harvested every 24 hours. Virus titers of EbolaδVP30-neo were determined in VeroVP30 cells, while virus titers of wild- type Ebolavirus were determined in wild-type Vero cells. To determine virus titers, cells were overlaid with 1.5% methylcellulose and 7 days later, assayed for VP40 expression using an immunostaining assay. EbolaδVP30-neo virus replicated efficiently in VeroVP30 cells at both conditions tested, reaching 10 ⁷ focal-forming units (FFU)/ml on day 6 postinfection (Figure 3, top panels, solid squares). No replication of EbolaδVP30-neo was detected in wild-type Vero cells (Figure 3, bottom panels, solid squares); the low titers that were detected for up to three days postinfection likely reflect input virus. Together, these findings attest to the biological confinement of the EbolaδVP30 system. The

replication kinetics of EbolaδVP30-neo in VeroVP30 cells are similar to those of wild-type Ebolavirus in either VeroVP30 (Figure 3, top panels, open circles) or wild-type Vero cells (Figure 3, bottom panels, open circles), establishing the described approach as a highly efficient method for generating biologically contained Ebolaviruses.

Morphology of EbolaδVP30-neo virus. Next, the morphology of EbolaδVP30-neo virus was assessed by transmission electron microscopy (TEM). VeroVP30 cells were infected with EbolaδVP30-neo virus and fixed 36 hours later. Samples were processed for TEM as described in Noda et al. (2002). As shown in Figure 4 (right panels), the particles budding from VeroVP30 cells infected with EbolaδVP30-neo virus were indistinguishable in their size and shape from wild-type Ebolaviruses (Figure 4, left panels). Thus, providing VP30 protein in trans does not have a discernable effect on virion morphology, suggesting that the described system would be suitable for studies of virion formation and budding, for example.

Taken together, the above results demonstrate that the EbolaδVP30-neo virus is biologically contained, replicates to high titers in a helper cell line, is genetically stable, and is morphologically indistinguishable from wild-type virions. Having provided proof-of-concept for the generation of biologically contained Ebolaviruses, the utility of this strategy in basic research and drug screening applications was assessed.

Generation of an EbolaδVP30-eGFP virus and its usefulness for basic research applications. An EbolaδVP30 virus encoding enhanced green fluorescence protein (eGFP) instead of VP30 was generated (Figure 1; designated EbolaδVP30-eGFP), using the same procedures described above for EbolaδVP30-neo virus. Analogous to EbolaδVP30-neo virus, the eGFP variant replicated efficiently with virus titers reaching 8.0 x 10 ⁷ FFU/mL. Expression of eGFP was observed as early as 10 hours postinfection (data not shown).

Takada et al. (2003) used replication-competent vesicular stomatitis virus (VSV) pseudotyped with Ebolavirus GP and two neutralizing monoclonal antibodies (mAb), 133/3.16 and 226/8.1, to map Ebolavirus GP epitopes and to generate escape mutants. To confirm with authentic Ebolavirus virions the findings of Takada et al. (2003) based on a VSV-pseudotyping system, escape mutants were generated by amplifying EbolaδVP30-eGFP virus in the presence

of mAb 133/3.16 or 226/8.1. Each of eight escape mutants to mAb 133/3.16 possessed a histidine-to-arginine substitution at position 549 (H549R) in GP, reported by Takada et al. (2003). Using mAb 226/8.1, 12 escape mutants were isolated that all contained an arginine-to-tryptophan substitution at position 134 (Rl 34W), a mutation identical to one identified by Takada et al. (2003).

However, the remaining two escape mutations described by Takada et al. (2003) were not detected. Whether this discrepancy in escape mutants reflects differences between the biological systems used or random mutations is presently unclear. Nonetheless, these experiments illustrated one of the ways that biologically contained Ebolaviruses could be used in basic research applications.

In conclusion, biologically contained Ebolaviruses lacking the VP30 gene afford a safe, alternative way to study authentic Ebolavirus, to develop Ebolavirus vaccines, and to screen chemical libraries for compounds that interfere with the Ebolavirus life cycle. Indeed, each of the three different biologically contained viruses generated (encoding neomycin or eGFP instead of VP30) was biologically contained, as demonstrated by their ability to replicate in VeroVP30 (a Vero cell line that stably expresses VP30 in trans), but not in wild- type Vero cells. Moreover, virus titers were in the range of 10 ⁷ FFU/mL and hence comparable to those obtained for wild-type Ebolavirus (Figure 3; Volchov et al., 2001; Neumann et al., 2002; Ebihara et al., 2006) while morphological, biochemical, and virological analyses indicated that the tested properties of EbolaδVP30 viruses were indistinguishable from those of wild-type Ebolavirus. These physical properties, together with the results of studies to illustrate the potential of biologically contained Ebolaviruses in basic research and drug screening applications, will greatly accelerate current filovirus research efforts.

Example 2

Ebola viruses (family Filoviridae), cause severe hemorrhagic fever in humans and nonhuman primates with mortality rates up to 90% (Johnson et al., 1977). Currently, there are no licensed vaccines or antivirals available against Ebola virus. A vaccine against Ebola virus is not only desirable for local populations in the epidemic areas of Africa, but also for health care workers during an outbreak and for post-exposure treatment of laboratory workers after

accidental exposure to the virus. A few vaccine candidates have been shown to protect mice, guinea pigs, or nonhuman primates against a lethal challenge of Ebola virus; however, each of these candidates has disadvantages, such as lack of protection in nonhuman primates, preexisting immunity against the vector in humans, or potential central nervous system involvement (Reed et al., 2007). Moreover, the current vaccine candidates are based on virus-like particles (VLPs) or virus- vectored vaccines, none of which express the full components of the viral antigens. On the other hand, the use of live attenuated vaccines may not be feasible for Ebola virus from a biosafety perspective. To overcome these potential limitations, biologically contained viruses offer an attractive option since they are biologically safe but provide all the viral antigens. Materials and Methods

Cells. VeroVP30 cells were established as described in Example 1 and grown in Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), L-glutamine, vitamins, non-essential amino acid solution, and 5μg/mL puromycin (Sigma, St. Louis, MO).

Viruses. The EbolaδVP30 virus was generated as described in Example 1. Briefly, using the plasmid containing the full-length Ebola cDNA genome of the Zaire Mayinga strain of Ebola virus (Neumann et al., 2002), the open reading frame (ORF) of VP30 was replaced with the ORF of the drug-resistant gene neomycin. Using Ebola virus reverse genetics (Neumann et al., 2002), the EbolaδVP30 virus was generated and passaged in a Vero cell line stably expressing VP30. EbolaδVP30 was propagated in VeroVP30 cells in MEM medium as described above, but supplemented with 2% FCS. The virus was harvested six days after infection of the cells at a multiplicity of infection (MOI) of 1 and directly stored at -80 ^° C. Harvested virus was also partially purified by ultracentrifugation at 27,000 rpm for 2 hours over 20% sucrose. The viral pellet was resuspended in sterile PBS and stored at -80°C. Viral titers were determined by plaque assay in confluent VeroVP30 cells overlaid with 2% FCS-MEM containing 1.5% methyl cellulose (Sigma).

Since wild-type Ebola virus does not kill mice, challenge studies were carried out with a mouse-adapted Ebola virus (Bray et al., 1998). This virus was generated as described in Ebihara et al., 2006 and used under BSL-4 conditions at the Canadian Centre for Human and Animal Health in Winnipeg, Canada.

Antibody titers. The levels of Ebola glycoprotein (GP)-specific immunoglobulin G (IgG) antibodies in vaccinated mice were examined by using an enzyme-linked immunosorbent assay (ELISA). Briefly, wells of Immulon 2HB plates (Thermon Labsystems, Franklin, MA) were coated with purified Ebola GP (Takada et al., 2001) and blocked with PBS containing 10 mg/mL bovine serum albumin. After incubation of Ebola GP-coated wells with mouse serum from control and vaccinated mice, bound antibodies were detected with goat anti-mouse IgG conjugated to horseradish peroxidase (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) by an ELISA plate reader at an absorbance of 405 nm.

Intracellular staining and flow cytometry. The number of cytokine- producing CD8 ⁺ T cells was determined by intracellular staining as described Murali-Krishna et al. (1998). Briefly, splenocytes were stimulated with the Ebola peptide NP _279-288 (SFKAALSSLA, derived from the nucleoprotein NP; SEQ ED NO:16) (Olinger et al., 2006; Simmons et al., 2004), VP40i ₇ i _-)80

(YFTFDLTALK, derived from the matrix protein VP40; SEQ ID NO: 17), or GPi _{6I-I 69} (LYDRLASTV, derived from GP) (Olinger et al., 2005; Warfield et al., 2005) for 5 hours in the presence of brefeldin A and IL-2. Following activation, cells were stained for cell surface CD8 ⁺ and intracellular EFNγby using the Cytofix/Cytoperm kit from BD Biosciences (San Jose, CA). The number of cytokine-producing CD8 ⁺ T cells was determined by using a FACSCalibur flow cytometer (BD Biosciences).

Vaccination and challenge. Four-week-old female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were anesthetized with isoflurane and intraperitoneally (D?) inoculated twice at three-week intervals with 10 ⁶ focus forming units (FFU) of sucrose-purified EbolaδVP30 virus (Figure 7); control mice were simultaneously inoculated with PBS. A second group of mice received three immunizations (at three- week intervals) with 10 ⁷ FFU of virus harvested from cell culture supernatant (Figure 7), or, as a control, 2% FCS- MEM. Vaccinations were conducted at the University of Wisconsin-Madison. Mice were then transported to the BSL-4 laboratory at the National Microbiology Laboratory of the Public Health Agency of Canada, where they were challenged with 1000 mouse lethal doses 50 (MLD ₅₀ ; i.e., the dose required to kill 50% of infected animals) of mouse-adapted Ebola virus. Four days after

challenge, viral titers were determined in the serum of three control and three vaccinated mice from each group. The remaining mice were monitored for survival for 28 days. All animal experiments were performed in accordance with approved animal use protocols and according to the guidelines set forth by the Canadian Council of Animal Care and the University of Wisconsin-Madison. Results

Antibody response of mice immunized with EbolaδVP30 virus. To assess the EbolaδVP30 virus as a potential vaccine, its immunogenicity in mice was determined. Mice vaccinated with the EbolaδVP30 virus did not show any signs of disease, demonstrating the lack of pathogenicity of the EbolaδVP30 virus. When serum samples, collected two weeks after each vaccination to determine the levels of antibodies to the Ebola glycoprotein (GP), were tested for IgG antibody by ELISA with purified GP (Figure 5), vaccinated animals showed elevated levels of antibody titers against GP after the first vaccination compared to control mice (Figure 5); these antibody titers further increased after the second and third vaccinations. This finding demonstrates the ability of the biologically contained EbolaδVP30 virus to elicit antibodies to GP.

CD8 ⁺ T-cell responses in vaccinated mice. The cellular response to vaccination in mice was examined. Mice were vaccinated as described above. Eight days after the second immunization, four vaccinated and two control mice were euthanized and their spleens removed. Splenocytes were isolated and stimulated with the Ebola peptide NP _279-288 (SFKAALSSLA), VP4O _17M8 o (YFTFDLTALK) or GPi _6M69 (LYDRLASTV) for 5 hours in the presence of brefeldin A and IL-2. Vaccinated mice had IFNγ-positive CD8 ⁺ cells in the range of 0.017% to 0.22% for cells stimulated with Ebola peptide NP _279-288 (Figure 6). For control mice, the number of IFNγ-positive CD8 ⁺ cells was significantly lower, ranging from 0.00513% to 0.00794% (Figure 6). No IFNγ- positive CD8 ⁺ cells were detected for cells stimulated with Ebola peptide VP4Oi _{7] -} i8o or GPi6i-i69 (data not shown). Protective efficacy of EbolaδVP30 virus in mice. To assess the protective efficacy of the EbolaδVP30 virus, two groups of 4-week-old mice were intraperitoneally immunized, then subjected to lethal challenge with mouse-adapted Ebola virus (Figure 7). 'Group 1' mice were immunized three times at three- week intervals with 10 ⁷ FFU of non-purified EbolaδVP30 virus

(i.e., virus harvested from cell culture supernatant); eight control mice were inoculated in the same manner with 2% FCS-MEM. Mice from this group were challenged seven weeks after the last immunization with 1000 MLD ₅₀ of mouse- adapted Ebola virus, which consistently kills mice (Bray et al., 1998; Ebihara et al., 2006). 'Group 2' mice were immunized twice (with a three-week interval) with 10 ⁶ FFU of purified EbolaδVP30 virus; ten control mice were similarly inoculated with PBS. Mice from 'Group 2' were challenged eight weeks after the last immunization with 1000 MLD ₅₀ of mouse-adapted Ebola virus. No signs of disease or illness were seen in mice vaccinated with purified or non-purified EbolaδVP30 virus, whereas control mice from both groups began showing signs of sickness (e.g., ruffled fur) along with weight loss on day 3 post-challenge (Figure 8a). By day 7 post-challenge, all control mice had succumbed to infection (Figure 8b). By contrast, vaccinated mice from both groups showed no signs of disease, as characterized by ruffled fur and weight loss (Figure 8a), and were fully protected against lethal challenge (Figure 8b) up to day 28, when all surviving mice were euthanized. On day 4 post-challenge, mice were sacrificed to determine viral titers in the sera (Figure 9). Vaccinated mice from both groups showed a 3 to 4 logi ₀ reduction in viral titers compared to their respective control mice. Taken together, these data demonstrate that the EbolaδVP30 virus efficiently protects mice against challenge with a lethal dose of mouse- adapted Ebola virus. Similar results were obtained in guinea pigs (Figures 11-13). Discussion

Here, it was demonstrated that EbolaδVP30-immunized mice were completely protected from a lethal challenge with mouse-adapted Ebola virus and that the virus titers in sera from these mice were more than 1000-fold lower than those in control mice. These results show the potential of this biologically contained Ebola virus as a vaccine.

The humoral response to Ebola virus infection is important, as demonstrated by protection from a lethal challenge by passive transfer of antibodies to the viral glycoprotein GP (Gupta et al., 2001 ; Warfield et al.,

2003). However, the ability of a vaccine to elicit an antibody response does not in itself correlate with protection from Ebola virus infection. For example, classical vaccine approaches, such as γ-irradiated Ebola and Marburg viruses, along with GP expressed in baculovirus generate a moderate antibody response;

however, they fail to protect mice against a lethal challenge (Ignatyeve et al., 1996; Lupton et al., 1980; Mellquist-Riemenschneider et al., 2003). By contrast, Ebola and Marburg VLPs protect mice from a lethal challenge of Ebola or Marburg virus (Warfield et al., 2003; Warfield et al., 2004; Warfield et al., 2005), and not only elicit a humoral response, but also induce a CD8 ⁺ T-cell response, highlighting the importance of the latter response for protection against a lethal challenge of Ebola virus (Warfield et al., 2005). Similarly, in non- human primates (NHPs), full protection from a lethal challenge appears to depend on both the humoral response and a CD8 ⁺ cellular response (Sullivan et al., 2000). Vaccine candidates that protect NHPs from a lethal Ebola virus challenge, such as recombinant vesicular stomatitis virus (VSV) (Jones et al., 2005) and adenovirus (Sullivan et al., 2000), induce a CD8 ⁺ T-cell response in NHPs, albeit to varying degrees (Jones et al., 2005; Sullivan et al., 2000). The EbolaδVP30 virus induced both humoral and CD8 ⁺ T-cell (specific for an Ebola NP epitope) responses, although the extent of the latter responses varied among animals (Figure 6). Whether this CD8 ⁺ T-cell response is sufficient to provide protection to NHPs from a lethal Ebola virus infection remains to be tested.

Although vaccine candidates such as recombinant VSV or parainfluenza virus offer protection in various animal models (Bukreyev et al., 2006; Jones et al., 2005), there are safety concerns with the use of these vaccines in humans (Bukreyev et al., 2006; Jones et al., 2005; Reed et al., 2007). Preexisting immunity to a vaccine based on recombinant adenovirus is also a concern, as is the large amount of virus (10 ¹⁰ particles) needed to confer protection in NHPs (Jones et al., 2005; Sullivan et al., 2000). Ebola and Marburg VLPs have been shown to protect mice and guinea pigs from a lethal challenge of these viruses (Warfield et al., 2004; Warfield et al., 2005). While VLPs are safe and, due to the rarity of Ebola virus infection, preexisting immunity to Ebola or Marburg viruses is not a concern for VLP vaccines, it is difficult to produce large quantities of VLPs from cell culture. The biologically contained EbolaδVP30 virus is thus an ideal vaccine candidate since it combines the advantages of VLPs and vectored vaccines (i.e., safety and efficacy), yet it can be propagated to high titers in VeroVP30 cells like standard viruses (Example 1). Further studies will include testing the EbolaδVP30 virus for its protective efficacy in NHPs. In addition, shorter,

single vaccination protocols will be evaluated to determine if the EbolaδVP30 virus vaccine could elicit fast and effective immunity in the event of an outbreak or bioterrorism attack. This includes evaluating the EbolaδVP30 virus as a vaccine for post-exposure treatment.

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All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.