Recombinant biologically contained filovirus vaccines

Abstract

To provide recombinant biologically contained filovirus and methods for producing and using these viruses.SOLUTION: Provided herein is a vaccine comprising an effective amount of a recombinant filovirus and one or more adjuvants, where the genome of the recombinant filovirus contains a deletion of one or more nucleotides in a polynucleotide sequence for a viral protein corresponding to Ebola virus VP30, and where the deletion is effective to prevent expression of a functional viral protein corresponding to Ebola virus VP30 upon infection of a cell with the recombinant filovirus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of the filing date of U.S. Patent No. 62 / 715,673, filed August 7, 2018, the disclosure of which is incorporated herein by reference.

[0002] [Federal Rights Statement] This invention was made with government support under A1109762 awarded by the National Institutes of Health. The federal government has certain rights in this invention. [Background technology]

[0003] Ebola virus causes hemorrhagic fever in humans and nonhuman primates, with a 90% case fatality rate in some outbreaks (Sanchez et al., 2007). Ebola virus and the closely related Marburg virus belong to the Filoviridae family (Feldman et al., 2004). Currently, there are no approved vaccines or antivirals for use against filoviruses, and no biosafety level-4 (BSL-4) containment is required for working with these viruses. The lack of sufficient BSL-4 space, trained personnel, and the rigors of working in a BSL-4 laboratory significantly hinder basic research with Ebola virus and vaccine development and large-scale screening for effective antiviral compounds. These limitations prompted the examination of various steps in the Ebola virus life cycle in the absence of infectious virus: (i) replication and transcription were studied by using reporter gene assays 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); (ii) entry and fusion processes were assessed by pseudotyping assays relying on the use of recombinant vesicular stomatitis viruses or retroviruses (Yonezawa et al., 2005; Wool-Lewis et al., 1998; Takada et al., 1997; Marzi et al., 2006); and (iii) budding was examined using virus-like particles generated from viral proteins provided by protein expression plasmids (Jasenosky et al., 2001; Licata et al., 2004; Noda et al., 2006). al., 2002; McCarthy et al., 2006; Johnson et al., 2006). However, some recent findings suggest that data obtained by these artificial mechanisms are not necessarily reproducible with live, authentic Ebola virus (Neumann et al., 2005).Therefore, there is a clear need for a biologically contained Ebola virus that resembles the wild-type virus but can be manipulated outside of BSL-4 containment. Summary of the Invention

[0004] The present invention provides a vaccine comprising an effective amount of a recombinant single-stranded, negative-stranded RNA virus, the genome of which contains a deletion of viral sequences corresponding to nonstructural or non-glycosylated viral proteins essential in trans for viral replication, and, in one embodiment, an insertion of one or more nucleotide sequences encoding one or more adjuvants, or, in one embodiment, one or more heterologous gene products, or, in one embodiment, an insertion of one or more nucleotide sequences encoding one or more adjuvants and one or more heterologous gene products, wherein the insertion can be within a coding or non-coding sequence. In one embodiment, the heterologous gene product is derived from a Zaire, Sudan, Côte d'Ivoire, Bundibugyo, Reston, or Marburg filovirus, or one or more glycoproteins of these filoviruses. In one embodiment, the insertion can replace a coding sequence, e.g., a glycoprotein coding sequence, or a portion thereof, or can replace a non-coding sequence. In one embodiment, the deletion is effective to inhibit or prevent viral replication upon infection of a cell with the recombinant single-stranded, negative-stranded RNA virus. For example, deletion of corresponding viral sequences in nonstructural or nonglycosylated viral proteins essential in trans for viral replication can be effective in preventing the expression of functional nonstructural or nonglycosylated proteins upon infection of cells by a recombinant negative-stranded single-stranded RNA virus. In one embodiment, deletion of corresponding viral sequences in nonstructural or nonglycosylated viral proteins essential in trans for viral replication can be within filovirus sequences in viral proteins corresponding to the Ebola virus VP30 protein. In one embodiment, the genome of the recombinant, biologically contained filovirus includes heterologous sequences, e.g., sequences associated with VP30, located within the deletion.Any deletion of viral sequences of a single-stranded negative-strand RNA virus can include deletion of one or more nucleotides, e.g., at least 0.1%, 1%, 5%, 10%, 50%, 60%, 70%, 80%, 90%, or any integer therebetween, and up to 100%, of viral sequences corresponding to nonstructural, glycosylated, or nonglycosylated viral proteins. Deletions of viral sequences corresponding to nonstructural or nonglycosylated viral proteins essential in trans for viral replication are stable over multiple passages and readily detectable, e.g., by RT-PCR. In one embodiment, the genome of the recombinant virus has deletions in viral sequences of two or more nonstructural or nonglycosylated proteins, e.g., deletions in sequences in viral proteins that are not adjacent to each other, e.g., deletions in sequences in viral proteins corresponding to the Ebola virus VP30 protein and the Ebola virus GP protein. In one embodiment, if the recombinant viral genome has a deletion in the viral sequence for a nonstructural, glycosylated, or nonglycosylated protein, at least a portion of the deleted viral sequence can be replaced with a nucleotide sequence encoding an antigen expressed in the recombinant filovirus that is a prophylactic or therapeutic agent when administered to a mammal. In one embodiment, if the recombinant viral genome has a deletion in the viral sequence for two or more proteins that are nonstructural, glycosylated, or nonglycosylated, at least a portion of the deleted viral sequence can be replaced with a nucleotide sequence encoding an antigen expressed in the recombinant filovirus that is a prophylactic or therapeutic agent when administered to a mammal. Vaccines of the invention can provide subtype cross-protection, filovirus cross-protection, and optionally as monovalent, bivalent, or multivalent vaccines in pathogens other than filoviruses.

[0005] As demonstrated herein below, incorporating an adjuvant into the vaccine provided unexpected results. Furthermore, not all adjuvants were effective; for example, aluminum did not enhance the efficacy of the vaccine virus of the present invention compared to a control (no adjuvant). In one embodiment, a monovalent recombinant filovirus vaccine comprises one or more adjuvants and a recombinant filovirus, expression of whose genome results in a virus with a homologous glycoprotein, e.g., the Zaire gene expresses the Zaire glycoprotein. In one embodiment, a monovalent recombinant filovirus vaccine comprises one or more adjuvants and a recombinant filovirus, expression of whose genome results in a virus with a heterologous glycoprotein, e.g., inserted into ORF4 (to replace the parental glycoprotein), e.g., the Zaire genome expresses the glycoprotein of Marburg virus, Sudan Ebola virus, or Bundibugyo Ebola virus. In one embodiment, a monovalent recombinant filovirus vaccine comprises one or more adjuvants and a recombinant filovirus, expression of whose genome results in a virus having a heterologous glycoprotein, e.g., the Zaire genome expressing a Marburg, Sudan, or Bundibugyo glycoprotein, e.g., inserted within a sequence corresponding to Ebola virus VP30 (ORF5) or resulting in a deletion of ORF5 or a portion thereof, e.g., so that two different glycoproteins are expressed.

[0006] In one embodiment, a vaccine is provided comprising a recombinant filovirus and an effective amount of one or more adjuvants. The genome of the recombinant filovirus contains a deletion of one or more nucleotides within a polynucleotide sequence for a viral protein corresponding to Ebola virus VP30, and the deletion is effective to prevent expression of a functional viral protein corresponding to Ebola virus VP30 upon infection of a cell with the recombinant filovirus. In one embodiment, at least 90% of the sequence corresponding to the VP30 sequence within the viral genome of the virus is deleted. In one embodiment, the genome further contains a nucleotide sequence encoding a heterologous prophylactic or therapeutic gene product. In one embodiment, the nucleotide sequence is inserted within 500 nucleotides of the site of the deletion or in the region of the deletion. In one embodiment, the nucleotide sequence is inserted within the filovirus genome outside the site of the deletion within the polynucleotide. In one embodiment, the nucleotide sequence is inserted between the NP coding sequence and the VP35 coding sequence within the filovirus genome. In one embodiment, the nucleotide sequence replaces a GP / sGP sequence or portion thereof. In one embodiment, the nucleotide sequence is inserted within the GP / sGP coding sequence. In one embodiment, the heterologous gene product comprises a heterologous filovirus glycoprotein. In one embodiment, the filovirus glycoprotein comprises a glycoprotein of Marburg virus, Ebola virus, Sudan virus, Tai Forest virus, Reston virus, or Bundibugyo virus. In one embodiment, the recombinant filovirus genome is a recombinant Ebola virus genome. In one embodiment, the additional vaccine comprises a pharmaceutically acceptable carrier.

[0007] In one embodiment, the adjuvant comprises lipopolysaccharide. In one embodiment, the lipopolysaccharide comprises monophosphoryl lipid A. In one embodiment, the adjuvant comprises squalene. In one embodiment, the adjuvant comprises Quillaja extract. In one embodiment, the adjuvant comprises saponin. In one embodiment, the recombinant filovirus in the vaccine is inactivated. Also provided is a method of immunizing a mammal with a composition comprising a recombinant filovirus. In one embodiment, the mammal is a human. In one embodiment, two doses of the composition are administered. In one embodiment, a single dose is administered. In one embodiment, three doses of the composition are administered. In one embodiment, the recombinant filovirus is inactivated using, for example, heat, one or more chemicals, such as formaldehyde, formalin, beta-propiolactone, diethylpyrocarbonate, an oxidizing agent, such as hydrogen peroxide, 2-2'-dithiodipyridine, binary ethyleneimine, glutaraldehyde, or radiation, such as gamma rays or UV rays.

[0008] Because most regions of Africa suffer from specific endemic or recurrent diseases, and the combinations vary by region, the present disclosure provides bivalent / multivalent vaccines that combat the combinations of diseases that impact specific regions. Monovalent vaccines may be particularly useful in response to any outbreaks that do not respond well to other vaccines. Multivalent vaccines may be based on the addition of exogenous sequences at any of several locations in the filovirus genome, including, but not limited to, 1) an artificial transcription unit between open reading frame (ORF) 1 (e.g., NP) and ORF 2 (e.g., VP35), 2) ORF 4 (e.g., the Zaire glycoprotein gene), and 3) ORF 5 (e.g., the VP30 gene). In one embodiment, the bivalent vaccine virus can express one or more non-glycosylated proteins, one or more glycosylated proteins, or at least one non-glycosylated protein and at least one glycosylated protein, e.g., from Zaire Ebola virus and Marburg virus, Ebola and Marburg virus, filovirus and Lassa virus, or filovirus and Plasmodium falciparum (malaria).

[0009] Thus, in one embodiment, the recombinant filovirus is a recombinant filovirus whose genome comprises a first deletion of one or more nucleotides in a viral protein polynucleotide sequence corresponding to Ebola virus VP30, the first deletion effective to prevent expression of a functional viral protein corresponding to Ebola virus VP30 upon infection of a cell with the recombinant filovirus, and whose genome encodes one or more filovirus glycoproteins. The genome may comprise a mutation in the region flanking the NP coding sequence and VP35 coding sequence, a mutation in the GP / sGP coding sequence, and / or an insertion within 500 nucleotides of the first site or at the site of the first deletion, or a combination thereof, and whose genome encodes one or more filovirus glycoproteins. The mutation in the region flanking the NP coding sequence and VP35 coding sequence comprises an insertion of a nucleotide sequence encoding a prophylactic or therapeutic heterologous gene product and, optionally, a deletion of one or more nucleotides in the region flanking the NP coding sequence and VP35 coding sequence. Mutations within the GP / sGP coding sequence include insertions of nucleotide sequences encoding prophylactic or therapeutic heterologous gene products and, optionally, deletions of one or more nucleotides within the GP / sGP coding sequence. Insertions within 500 nucleotides of or at the site of the first deletion encode prophylactic or therapeutic heterologous gene products. In one embodiment, the recombinant filovirus is inactivated using, for example, heat, one or more chemicals, such as formaldehyde, formalin, beta-propiolactone, diethylpyrocarbonate, oxidizing agents, such as hydrogen peroxide, 2-2'-dithiodipyridine, binary ethyleneimine, glutaraldehyde, or irradiation, such as gamma rays or UV rays.

[0010] Also provided is a multivalent vaccine comprising an effective amount of a recombinant filovirus, wherein the genome of the recombinant filovirus comprises a first deletion of one or more nucleotides in a viral protein polynucleotide sequence corresponding to Ebola virus VP30, the deletion effective to inhibit expression of a functional viral protein corresponding to Ebola virus VP30 upon infection of a cell with the recombinant filovirus, and wherein the genome encodes one or more filovirus glycoproteins. The genome may comprise a mutation in the regions flanking the NP and VP35 coding sequences, a mutation in the GP / sGP coding sequence, and / or an insertion within 500 nucleotides of or at the site of the first deletion, or a combination thereof. The mutation in the regions flanking the NP and VP35 coding sequences may comprise an insertion of a nucleotide sequence encoding a prophylactic or therapeutic heterologous gene product, and also optionally a deletion of one or more nucleotides in the regions flanking the NP and VP35 coding sequences. The mutation in the GP / sGP coding sequence includes the insertion of a nucleotide sequence for a heterologous prophylactic or therapeutic gene product and, optionally, the deletion of one or more nucleotides within the GP / sGP coding sequence. The insertion, within 500 nucleotides, e.g., at least 1000 nucleotides, of the first deletion site or at the first deletion site, encodes a heterologous prophylactic or therapeutic gene product. In one embodiment, one filovirus glycoprotein encoded by the genome comprises a glycoprotein of a homologous filovirus. In one embodiment, one filovirus glycoprotein encoded by the genome comprises a glycoprotein of a heterologous filovirus. In one embodiment, the heterologous prophylactic or therapeutic gene product is not a glycoprotein. In one embodiment, the heterologous prophylactic or therapeutic gene product comprises a glycoprotein. In one embodiment, the vaccine further comprises an adjuvant. In one embodiment, the adjuvant comprises lipopolysaccharide. In one embodiment, the adjuvant comprises squalene. In one embodiment, the adjuvant comprises a Quillaja extract. In one embodiment, the adjuvant comprises a saponin. In one embodiment, the vaccine further comprises a pharmaceutically acceptable carrier.In one embodiment, the recombinant filovirus in the vaccine is inactivated. Also provided is a method of immunizing a mammal, e.g., a human, by administering to the mammal an effective amount of the vaccine. For example, a human who has come into contact with a filovirus-infected individual or who has been inadvertently exposed to a filovirus, e.g., in a laboratory, can be administered a recombinant, infectious, biologically contained virus of the invention in an amount effective to inhibit or persistently eliminate filovirus replication in the human.

[0011] To prepare such a virus, we utilized reverse genetics in negative-strand RNA viruses to generate Ebola viruses lacking substantial portions of the VP30 gene (encoding an essential transcription factor), lacking substantial portions of its L gene, or lacking substantial portions of both genes, designated EbolaΔVP30 viruses. The EbolaΔVP30 viruses were genetically stably maintained and biologically restricted to cell lines expressing VP30. Thus, the EbolaΔVP30 viruses fulfill several criteria for vaccine viruses: they can be propagated to reasonably high titers in helper cells, are genetically stable (as determined by sequence analysis after seven serial passages in VP30-expressing Vero cells), and are safe. Furthermore, the resulting viruses appear wild-type in their life cycle, morphology, and growth characteristics but can be handled in non-BSL-4 laboratories, opening new opportunities for the study of the Ebola virus life cycle and the identification of effective antiviral compounds.

[0012] Other single-stranded negative-strand RNA viruses can be similarly engineered, such as Nipah, Hendra, and Henipaviruses, to mutate or delete their corresponding sequences in nonstructural or nonglycosylated viral proteins required for viral replication. Thus, the genomes of viruses in the following families can be engineered to provide infectious, biologically contained viruses that resemble wild-type viruses in their life cycle, morphology, and growth characteristics, can be grown to reasonably high titers in helper cells, are genetically stable, and are safe: Bornaviridae, Rhabdoviridae, Filoviridae (Marburg and Ebolavirus genera), Paramyxoviridae, Avulaviruses, Henipaviruses, Morbilliviruses, Respiroviruses, or Rubulaviruses.

[0013] The present disclosure also provides a method for preparing an infectious, biologically contained, single-stranded, negative-sense RNA virus, e.g., a filovirus. In one embodiment, the method includes providing a host cell, e.g., a Vero cell, with a plurality of viral vectors that, when expressed (stably or transiently), result in an infectious, biologically contained, single-stranded, negative-sense RNA virus. In one embodiment, the plurality of vectors includes a vector for vRNA production comprising a promoter operably linked to viral DNA, the viral DNA comprising a deletion of a sequence in the viral gene corresponding to Ebola virus VP30, the deletion being linked to a transcription termination sequence and an insertion of a heterologous sequence as discussed above, effective to inhibit expression of a functional viral protein corresponding to Ebola virus VP30. The host cell also includes a vector for mRNA production comprising a promoter operably linked to a DNA fragment encoding a viral polymerase, a DNA fragment encoding one or more other viral proteins, the viral proteins being viral proteins required for viral replication along with the viral polymerase and nucleoprotein, and a vector comprising a promoter operably linked to DNA encoding an RNA polymerase heterologous to the host cell. The heterologous RNA polymerase is selected to drive transcription of viral DNA containing deletions. In one embodiment, the vector for vRNA contains a T7 polymerase promoter and a ribozyme sequence capable of cleaving the transcript and resulting in a vRNA-like 3' end. Infectious, biologically contained virus is then isolated from the cells. In one embodiment, the host cells are transiently transfected with multiple vectors, and virus is harvested within 1, 2, 3, and up to 7 days after transfection. In one embodiment, the host is one approved for vaccine production. In one embodiment, additional heterologous sequences are contained in the vRNA vector or mRNA vector, subsequently introduced into the host cell and / or introduced into the host cell via an mRNA vector. In one embodiment, the additional heterologous sequence is an immunogenic polypeptide or peptide of a pathogen, a cancer antigen, or a therapeutic protein.

[0014] In one embodiment, a method for preparing a multivalent infectious, biologically contained filovirus is provided, which method is effective to produce an infectious, biologically contained filovirus when expressed in a host cell, wherein the vectors include a vector for vRNA production comprising a promoter operably linked to filovirus DNA containing a deletion in a functional viral protein sequence corresponding to Ebola virus VP30, where the deletion is a transcription termination sequence and other sequences as described herein above, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding a filovirus nucleoprotein, a filovirus corresponding to Ebola virus VP30, and a vector for mRNA production comprising a promoter operably linked to a filovirus DNA segment encoding a filovirus nucleoprotein. The methods include vectors for producing mRNA comprising a promoter operably linked to a DNA fragment encoding a filovirus protein, vectors for producing mRNA comprising a promoter operably linked to a DNA fragment encoding a filovirus protein corresponding to Ebola virus VP35, and vectors for producing mRNA comprising a promoter operably linked to DNA encoding an RNA polymerase heterologous to a host cell, wherein the heterologous RNA polymerase promotes transcription of vRNA from the filovirus DNA containing the deletion; and isolating infectious, biologically contained filovirus from the host cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a primate cell. In one embodiment, the cell is a Vero cell. In one embodiment, the heterologous RNA polymerase is T3, T7, or SP6 polymerase. In one embodiment, the gene product sequence encodes an immunogenic polypeptide or peptide of a pathogen, a cancer antigen, or a therapeutic protein. In one embodiment, each vector encoding a filovirus protein is on a separate plasmid.

[0015] Also provided is a method for producing, e.g., large-scale production of a recombinant filovirus, e.g., a vaccine product. The method includes culturing mammalian cells expressing a recombinant filovirus genome in serum-free medium in a cell culture system to produce progeny recombinant, biologically contained filoviruses. The recombinant filovirus genome includes a deletion of one or more nucleotides in a polynucleotide sequence in a viral protein corresponding to Ebola virus VP30, and the deletion is effective to inhibit expression of a functional viral protein corresponding to Ebola virus VP30 upon infection of the cells with the recombinant filovirus. In one embodiment, the serum-free medium includes a non-animal protein or peptide, e.g., a plant protein or peptide, an ion chelator, e.g., EDTA, ferric nitrate, ferrous sulfate, or transferrin, or a combination thereof. Supernatant from the progeny-bearing mammalian cells is collected and contacted with DNase and a virus-inactivating agent, e.g., beta-propiolactone, heat, formaldehyde, gamma radiation, or hydroxylamine, thereby providing an inactivated virus preparation. The inactivated virus preparation is subsequently purified, concentrated, desalted, and / or fractionated from other molecules, e.g., via filtration, under conditions that do not result in precipitation of inactivated virus particles, e.g., optionally visible to the naked eye.

[0016] In one embodiment, the mammalian cells are Vero cells. In one embodiment, the collected supernatant undergoes separation, such as filtration, before contact with DNase or the virus inactivating agent. In one embodiment, the collected supernatant is filtered through a 0.5 to 5 micron filter or a 1 to 5 micron filter. In one embodiment, the collected supernatant is filtered through a 0.01 to 1 micron filter or a 0.05 to 0.25 micron filter. In one embodiment, the inactivated virus preparation is filtered through a 0.01 to 1 micron filter or a 0.05 to 0.25 micron filter. In one embodiment, the inactivated virus preparation is combined with one or more adjuvants. [Brief explanation of the drawings]

[0017] [Figure 1] Schematic of the EbolaΔVP30 construct. Schematic of the Ebola virus genome flanked by the leader (l) and trailer (t) sequences in the positive-sense orientation (top row). Two unique restriction enzyme sites, SalI and SacI (positions 6180 and 10942 in the viral antigenome, respectively), allowed for the subcloning of a fragment spanning the VP30 gene. This subgenomic fragment was subsequently used to replace the VP30 gene with genes encoding neomycin (neo) or enhanced green fluorescent protein (eGFP), respectively. The unique restriction enzyme sites were used to amplify the altered subgenomic fragment back into a full-length Ebola virus cDNA construct.

[0018] [Figure 2] Representative filovirus sequences (Accession Nos. NC006432, NC004161, AY769362, AY142960, AF522874, AF499101, L11365, NC001608, 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). DETAILED DESCRIPTION OF THE INVENTION

[0019] definition A "vector" or "construct" (sometimes referred to as a gene delivery or gene transfer "vehicle") refers to a macromolecule or molecular complex comprising a polynucleotide or virus that is delivered to a host cell, either in vitro or in vivo. The delivered polynucleotide or virus may contain a coding sequence of interest for gene therapy. Vectors include, for example, viral vectors (e.g., filoviruses, adenoviruses, adeno-associated viruses (AAV), lentiviruses, herpesviruses, and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes that can mediate delivery of a polynucleotide to a host. Vectors may also contain other components or functionalities that further modulate gene delivery and / or gene expression, or otherwise provide advantageous properties to the target cell. Such other components include, for example, components that affect cell binding or targeting (including components that mediate cell-type or tissue-specific binding); components that affect uptake of vector nucleic acid by cells; components that affect localization of the polynucleotide within the cell after uptake (such as agents that mediate nuclear localization); and components that affect expression of the polynucleotide. Such components are also contemplated to include markers, e.g., detectable and / or selectable markers that can be used to detect or select for cells that have taken up and express the nucleic acid delivered by the vector. Such components can be provided as natural features of the vector (such as the use of certain viral vectors that have components or functionality that mediate binding and uptake), or the vector can be engineered to provide such functionality. A wide variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, it can either be stably replicated by the cell during mitosis as an autonomous structure, integrated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0020] "Recombinant viral vector" refers to a viral vector containing one or more modifications, including deletions, insertions, and / or heterologous genes or sequences. Because many viral vectors exhibit size limitations associated with packaging, the heterologous gene or sequence is typically introduced by replacing one or more portions of the viral genome. Such viruses can be replication-deficient (biologically contained) and require the deleted functions to be provided in trans (e.g., by using a helper virus or packaging cell line carrying the necessary genes for replication and / or encapsidation). Modified viral vectors in which the polynucleotide to be delivered is carried on the outside of the viral particle have also been described.

[0021] As used herein, the terms "gene delivery," "gene transfer," and the like (sometimes referred to as "gene transfer") refer to the introduction of an exogenous polynucleotide into a host cell, regardless of the method used for that introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., via viral infection / transfection or various other protein-based or lipid-based gene delivery complexes) and techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques for polynucleotide introduction). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contain an origin of replication compatible with the host cell or integrate into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. Many vectors are known in the art to mediate gene transfer into mammalian cells.

[0022] By "transgene" is meant any nucleic acid molecule (e.g., DNA) that is inserted transiently or permanently into a cell by any strategy and becomes part of the tissue when integrated into the genome or maintained extrachromosomally. Such transgenes may include the insertion of at least a portion of an open reading frame of a heterologous (i.e., foreign) gene, either partially or entirely, into an endogenous gene in the tissue, which portion optionally encodes a polypeptide having substantially the same activity as the corresponding full-length polypeptide or at least one activity of the corresponding full-length polypeptide.

[0023] By "transgenic cells" is meant cells that contain a gene. For example, cells stably or transiently transformed with a vector containing an expression cassette are transgenic cells that can be used to generate a population of cells having altered phenotypic characteristics. A "recombinant cell" is one that has been genetically modified, e.g., by genetic manipulation to insert, delete, or replace sequences within a non-recombinant cell.

[0024] The terms "wild-type" or "native" refer 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 therefore arbitrarily designated the "normal" or "wild-type" form of that gene. In contrast, "modified" or "mutant" refers to a gene or gene product that exhibits modifications in sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is known that naturally occurring mutations can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0025] The term "transduction" refers to the delivery of a polynucleotide to a recipient cell either in vivo or in vitro via a viral vector, and preferably a replication-deficient viral vector.

[0026] The term "heterologous," as it relates to nucleic acid sequences, e.g., gene sequences encoding proteins and regulatory sequences, refers to sequences that are not normally linked together and / or normally associated with a particular cell, e.g., from different origins (e.g., 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 vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in that context in nature. For example, a heterologous region of a nucleic acid construct would be considered to include a sequence not found in context with the coding sequence in nature, i.e., a coding sequence adjacent to a heterologous promoter. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from the native gene). Similarly, cells transformed with a construct that is not normally present are considered heterologous for purposes of the present invention.

[0027] "DNA" refers to the polymeric form derived from deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in double- or single-stranded form found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of a particular DNA molecule, the sequence will be described herein according to the usual convention of providing only the sequence in the 5' to 3' direction along the non-transcribed DNA strand (i.e., the strand having the sequence complementary to the mRNA). The term capture molecule includes molecules containing the four bases adenine, guanine, thymine, or cytosine, as well as base analogs known in the art.

[0028] 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 "AGT" is complementary to the sequence "TCA." Complementarity can be "partial," where only the bases of certain nucleic acids are matched according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between 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 and detection methods that depend on binding between nucleic acids.

[0029] DNA molecules are considered to have 5' and 3' ends because mononucleotides are derived from oligonucleotides or polynucleotides in such a way that the 5' phosphate group of one mononucleotide pentose ring is attached in one direction to the 3' oxygen atom of its neighbor via a phosphodiester bond. Therefore, an end of an oligonucleotide or polynucleotide is referred to as its 5' end when its 5' phosphate group is not linked to the 3' oxygen of a mononucleotide pentose ring, and as its 3' end when its 3' oxygen is not linked to the 5' phosphate of a consecutive mononucleotide pentose ring. As used herein, nucleic acid sequences, even if internal to a larger oligonucleotide or polynucleotide, can also be considered to have 5' and 3' ends. In either a linear or circular DNA molecule, distinct elements are referred to as being "upstream" or 5' of "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' manner along the DNA strand. Promoter and enhancer elements that direct transcription of linked genes are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effects 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.

[0030] A "gene," "polynucleotide," "coding region," "sequence," "fragment," or "transgene" that "encodes" a particular protein is a nucleic acid molecule that is transcribed and, optionally, 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 in either cDNA, genomic DNA, or RNA form. If in 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 includes, 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 often be located 3' to the gene sequence.

[0031] 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 transcription of a coding sequence in a recipient cell. Not all of these control elements will always be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0032] As used herein in its conventional sense, the term "promoter" refers to a nucleotide region containing DNA regulatory sequences derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence.

[0033] By "enhancer" is meant a nucleic acid sequence that, when positioned proximal to a promoter, confers increased transcriptional activity relative to the transcriptional activity resulting from the promoter in the absence of the enhancer domain.

[0034] With reference to nucleic acid molecules, "operably linked" means that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and an enhancer element) are linked in a manner that allows for transcription of the nucleic acid molecules. With reference to peptide and / or polypeptide molecules, "operably linked" means that two or more peptide and / or polypeptide molecules are linked in a manner that results in 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.

[0035] "Homology" refers to the degree of identity between two polynucleotides or two polypeptides. The correspondence between one sequence and another can be determined by techniques known in the art. For example, homology can be determined by direct comparison of the sequence information between two polypeptide molecules by coordinating the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that result in the formation of stable duplexes between homologous regions, followed by digestion with a single-strand-specific nuclease and sizing of the digested fragments. As determined using the above methods, two DNA or two polypeptide sequences are "substantially homologous" to each other if 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 the defined length of the molecule.

[0036] By "mammal" is meant any member of the Mammalia, including, but not limited to, humans and non-human primates, e.g., chimpanzees and other ape and monkey species; farm animals, e.g., cattle, sheep, pigs, goats, and horses; domestic animals, e.g., dogs and cats; laboratory animals, including rodents, e.g., mice, rats, rabbits, and guinea pigs.

[0037] By "derived" it is meant that a nucleic acid molecule is either made or designed from a parent nucleic acid molecule, and the derivative retains substantially the same functional characteristics of the parent nucleic acid molecule, e.g., encodes a gene product that has substantially the same activity as the gene product encoded by the parent nucleic acid molecule from which it is made or designed.

[0038] By "expression construct" or "expression cassette" is meant a nucleic acid molecule capable of directing transcription. An expression construct includes at least a promoter. Additional elements, such as enhancers, and / or transcription termination signals, may be included.

[0039] The term "exogenous," when used with reference to a protein, gene, nucleic acid, or polynucleotide in a cell or tissue, means a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or tissue by artificial or natural means. An exogenous nucleic acid can be derived from a different tissue or cell, or can be a copy of one or more additional nucleic acids that naturally occur in the tissue or cell. As a non-limiting example, an exogenous nucleic acid in a chromosomal location different from that of the native cell, or otherwise flanked by different nucleic acid sequences found in nature.

[0040] The term "isolated," when used with respect to a nucleic acid, peptide, polypeptide, or virus, means a nucleic acid sequence, peptide, polypeptide, or virus that is free from association with at least one contaminating nucleic acid, polypeptide, e.g., material in vivo, or substantially purified from material in vitro, such that it is identified and separated from other biological components, including those commonly associated with its natural source. An isolated nucleic acid, peptide, polypeptide, or virus exists in a form or configuration that differs from that in which it is found in nature. For example, a given DNA sequence (e.g., a gene) is found on a host chromosome adjacent to neighboring genes; an RNA sequence, e.g., a particular mRNA sequence encoding a particular protein, is found within a cell as a mixture with many other mRNAs encoding many other proteins. The isolated nucleic acid molecule can exist in single-stranded or double-stranded form. When the isolated nucleic acid molecule is used to express a protein, the molecule will contain at least the sense or coding strand (i.e., the molecule can be single-stranded), but can contain both the sense and antisense strands (i.e., the molecule can be double-stranded).

[0041] As used herein, the terms "recombinant nucleic acid" or "recombinant DNA sequence, molecule, or fragment" refer to nucleic acid, e.g., DNA, derived or isolated from a source that can be subsequently chemically altered in vitro, including, but not limited to, naturally occurring, non-naturally occurring, or sequences corresponding to naturally occurring sequences that are not located as they would be located in a natural genome. An example of DNA "derived" from a source would be a DNA sequence that is defined as a useful fragment and subsequently essentially only that material is chemically synthesized. An example of such DNA "isolated" from a source would be a useful DNA sequence that is deleted or removed from the source by chemical means, e.g., by the use of restriction endonucleases, or, in the context of the present invention, by genetic engineering methodologies, e.g., amplification, so that it can be further manipulated.

[0042] As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule that is expressed from a recombinant DNA molecule.

[0043] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein unless otherwise distinguished.

[0044] The term "sequence homology" refers to the percentage of base matches between two nucleic acid sequences or the percentage of amino acid matches between two amino acid sequences. Sequence homology is expressed as a percentage, e.g., 50%, which indicates the percentage of matches over the length of a selected sequence compared to some other sequence. Gaps (in either of the two sequences) are allowed to maximize matching; gap lengths of 15 bases or less are often used, preferably 6 bases or less, and more preferably 2 bases or less. When using oligonucleotides as probes or therapeutics, the sequence homology between a target nucleic acid and its oligonucleotide sequence is generally 17 or more target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably 9 or more matches out of 10 possible base pair matches (90%), and more preferably 19 or more matches out of 20 possible base pair matches (95%).

[0045] The term "selectively hybridize" means to detectably and specifically bind. The polynucleotides, oligonucleotides, and fragments of the present invention selectively hybridize to nucleic acid strands under hybridization and wash steps that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions are known in the art and can be used to achieve selective hybridization conditions as discussed herein. Generally, nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the present invention and the nucleic acid sequence of interest is at least 65%, and more typically has increasing homology of preferably at least about 70%, about 90%, about 95%, about 98%, and 100%.

[0046] Two amino acid sequences are homologous if there is 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 to maximize matching; gap lengths of 5 or less are preferred, and 2 or less are more preferred. Alternatively, and preferably, two protein sequences (or polypeptide sequences derived therefrom that are at least 30 amino acids in length) are homologous, as that term is used herein, if they have an alignment score of 5 or greater (in standard deviation units) using the ALIGN program with a mutation data matrix and a gap penalty of 6 or greater. Two sequences or portions thereof, when aligned using the ALIGN program, are more preferably homologous if 50% or more of their amino acids are the same, if desired.

[0047] As used herein, the term "corresponding" means that a polynucleotide sequence is homologous (e.g., identical, although not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence encoding a polypeptide or its complement, or that a polypeptide sequence is identical in sequence or function to a reference polypeptide sequence. By way of example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0048] The following terms are used to describe sequence correlation between two or more polynucleotides: "reference sequence," "comparison window," "sequence homology," "percentage of sequence homology," and "substantial homology." A "reference sequence" is a defined sequence used as a basis for sequence comparison; a reference sequence can be a subset of a longer sequence, such as a fragment of a full-length cDNA or gene sequence given in a sequence listing, or can include the complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, often at least 25 nucleotides in length, and frequently at least 50 nucleotides in length. Two polynucleotides each contain (1) sequences that are similar between the two polynucleotides (i.e., portions of the complete polynucleotide sequence), and (2) sequences that differ between the two polynucleotides. Sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare partial regions of sequence similarity.

[0049] As used herein, a "comparison window" refers to a conceptual segment of at least 20 contiguous nucleotides, where the portion of a polynucleotide sequence within the comparison window may contain no more than 20 percent additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Sequence alignment to determine the optimal comparison window can be performed by using a partial homology algorithm or by searching for similar methods, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA Genetics Software Package), or by inspection, and selecting the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) produced by the various methods.

[0050] The term "sequence homology" means that two polynucleotide sequences are identical over a window of comparison (i.e., on a nucleotide-by-nucleotide basis). The term "percentage of sequence homology" means that two polynucleotide sequences are identical over a window of comparison (i.e., on a nucleotide-by-nucleotide basis). The term "percentage of sequence homology" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions with identical nucleobases (e.g., A, T, C, G, U, or I) that occur in both sequences, resulting in the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., window size), and multiplying the result by 100 to yield the percentage of sequence homology. As used herein, the term "substantial homology" refers to a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 85 percent sequence identity, preferably at least 90-95 percent sequence identity, and more often at least 99 percent sequence identity, compared to a reference sequence over a comparison window of at least 20 nucleotide positions, often over a window of at least 20-50 nucleotides, where the percentage of sequence identity is calculated by comparing the polynucleotide sequence to the reference sequence, which may include a combined deletion or addition of up to 20 percent of the reference sequence over the comparison window.

[0051] As applied to polypeptides, the term "substantial homology" means two peptide sequences, when optimally aligned, e.g., by the GAP or BESTFIT programs, using default gap weights, share at least about 80% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 95% sequence identity, and most preferably at least about 99% sequence identity.

[0052] "Protective immune response" and "prophylactic immune response" are used interchangeably and refer to an immune response that targets immunogens to which an individual has not been exposed, or that targets proteins associated with disease in disease-free individuals, e.g., cancer-associated proteins in cancer-free patients.

[0053] "Therapeutic immune response" means an immune response that targets immunogens to which an individual has not been exposed or proteins associated with a disease in an individual with that disease.

[0054] The term "prophylactically effective amount" is intended to mean the amount necessary to prevent an individual from developing an infection, in the case of an infectious agent, and to prevent an individual from developing a disease, in the case of a disease.

[0055] The term "therapeutically effective amount" is intended to mean the amount necessary to reduce the level of infection in an infected individual to reduce symptoms or eliminate the infection, in the case of an infectious agent, and to reduce symptoms or treat the individual, in the case of a disease.

[0056] "Reducing an immune response to an immunogen" is meant to refer to the induction of an immune response in naive individuals and in individuals previously exposed to an immunogen, where the immune response to the immunogen is enhanced.

[0057] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition in which the target species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will contain about 80 percent or more of all macromolecular species present in the composition, more preferably about 85%, about 90%, about 95%, and about 99%. Most preferably, the target species is purified to the requisite homogeneity (contaminant species in the composition cannot be detected by conventional detection methods) and wherein the composition consists essentially of a single macromolecular species.

[0058] As used herein, "transfected," "transformed," or "transduced" includes any host cell or cell line that has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells of the invention are typically produced by transfection with a DNA sequence, such as an isolated linear DNA sequence, in a plasmid expression vector, or by infection with a recombinant viral vector.

[0059] Exemplary Viruses and Methods of the Invention The present invention provides isolated vectors, e.g., plasmids encoding single-stranded negative-strand RNA viral proteins and / or expressing vRNA from recombinant nucleic acids corresponding to sequences in mutant single-stranded negative-strand RNA viruses. When introduced into cells, the combination of these vectors can generate recombinant, infectious, biologically contained viruses. Thus, the present invention includes host cells that produce the recombinant, infectious, biologically contained viruses of the present invention. In one embodiment, the present invention provides isolated vectors, e.g., plasmids encoding filovirus proteins and / or expressing mutant filovirus vRNA, that, when introduced into cells, can result in recombinant, infectious, biologically contained filoviruses. The present invention also includes helper cells prepared by the methods disclosed herein, and host cells that transiently or stably produce recombinant, infectious, biologically contained filoviruses, including isolated recombinant filoviruses.

[0060] Vectors of the invention include vectors for mRNA production and vRNA production. In one embodiment, the vectors contain filovirus DNA, e.g., vectors for mRNA production having sequences corresponding to one or more open reading frames encoding filovirus proteins, or vectors for vRNA production containing deletions of full-length genomic sequences, including deletions of internal filovirus sequences corresponding to portions of at least one open reading frame. RNA produced by vRNA vectors can be packaged into viral particles in the presence of filovirus proteins, but as part of the resulting viral particles, it cannot replicate and does not result in virus production when introduced into cells that otherwise support filovirus replication, and the cells do not express at least one filovirus protein in trans, e.g., in cells that are not filovirus helper cells.

[0061] For example, Ebola virus has a negative-stranded, non-segmented RNA genome approximately 19 kilobases long that encodes seven structural proteins and at least one nonstructural 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 a matrix protein involved in virus budding (Harty et al., 2000; Panchal et al., 2003). VP24 is involved in the formation of the nucleocapsid, which is composed of NP, VP35, and viral RNA (Huang et al., 2002). Of the viral surface glycoproteins, only the 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 / insertion and optional replacement with heterologous sequences include, but are not limited to, sequences corresponding to the Ebola virus VP30 sequence or other single-stranded negative-strand RNA viruses, e.g., sequences of nonstructural, non-polymerase, and / or non-glycosylated viral proteins, or sequences within non-coding regions. The vectors can also contain genes or portions thereof other than the vector of a single-stranded negative-strand RNA virus, such as filovirus (heterologous sequences), with the aim of expressing the genes or portions in the host cell, either as proteins or by incorporation into vRNA. Thus, the vectors of the invention can contain additional viral sequences, e.g., filovirus sequences, genes or open reading frames of interest, e.g., heterologous genes for useful immunogenic peptides or proteins, such as vaccines or therapeutic proteins.

[0062] When more than one vector is used, the vectors can be physically linked, or each vector can be present in an individual plasmid or other, e.g., linear, nucleic acid delivery vehicle. The vector or plasmid can be introduced into any host cell, e.g., a eukaryotic cell that supports viral replication, e.g., a mammalian cell. Host cells useful for preparing the viruses of the invention include, but are not limited to, insect, avian, or mammalian host cells, e.g., canine, feline, equine, bovine, ovine, or primate cells, including simian or human cells. In one embodiment, the host cell is approved for vaccine production.

[0063] Viruses produced by the methods described herein are useful in viral mutagenicity studies, drug screening, and vaccine production (e.g., for AIDS, influenza, hepatitis B, hepatitis C, rhinovirus, filovirus, malaria, herpes, and foot-and-mouth disease), as well as gene therapy vectors (e.g., for cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamylase deficiency, and central nervous system tumors). In particular, the infectious, biologically contained filoviruses of the invention that induce strong humoral and cellular immunity can be used as vaccine vectors because they are unlikely to generate infectious recombinant viruses.

[0064] Thus, viruses for use in medical therapy (e.g., for vaccines or gene therapy) are provided. For example, the present invention provides a method of immunizing an animal against a pathogen, e.g., a microorganism, a virus such as Ebola virus, or a parasite, or a malignant tumor. The method comprises administering to the animal an effective amount of an isolated virus of the present invention that encodes and expresses at least one nucleic acid for an immunogenic peptide or protein of the pathogen or tumor effective to immunize the animal, optionally in combination with an adjuvant.

[0065] For the preparation of expression cassettes for transformation according to the present invention, the recombinant DNA sequence or fragment can be circular or linear, double-stranded or single-stranded. A DNA sequence encoding an RNA sequence substantially complementary to an mRNA sequence encoding a gene product of interest is typically a "plus" DNA sequence cloned into the cassette in the opposite orientation (i.e., 3-to-5 rather than 5-to-3). Also, the DNA sequence or fragment is generally in the form of chimeric DNA, such as plasmid DNA, which may contain coding regions flanked by control sequences that facilitate expression of the DNA in cells. As used herein, "chimeric" means that the vector contains DNA from at least two different species, or from the same species, linked or associated in a way that does not occur "naturally" or in the wild-type species.

[0066] In addition to DNA sequences that contribute as transcription units, or portions thereof, portions of DNA can be non-transcribed and contribute regulatory or structural functions. For example, the DNA can itself contain a promoter that is active in eukaryotic cells, e.g., mammalian cells, or in a cell type, or can utilize a promoter already present in the genome that is the target of lymphotropic viral transformation. Such promoters include the CMV promoter, the SV40 late promoter, and retroviral LTRs (long terminal repeats), such as those of MMTV, RSV, MLV, or HIV, although many other promoter sequences known in the art can be used in the practice of the invention.

[0067] Other elements functional in the host cell may also be part of the recombinant DNA, e.g., introns, enhancers, polyadenylation sequences, etc. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by influencing transcription, mRNA stability, etc. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the cell.

[0068] The recombinant DNA introduced into the cells may contain a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of transformed cells from the population of cells selected for transformation. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation step. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable their expression in the host cell. Useful selectable markers are well known in the art and include, for example, drug resistance genes and herbicide resistance genes, such as neo, hpt, dhfr, bar, aroA, puro, hyg, dapA, and the like. These genes are also listed in Table 1 of Lundquist et al. (U.S. Patent No. 5,848,956).

[0069] Reporter genes were used to identify potentially transformed cells and evaluate the functionality of regulatory sequences. Reporter genes that encode easily analyzable proteins are well known in the art. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organ or tissue, and it encodes a protein whose expression is manifested by some easily detectable property, such as enzymatic activity. Exemplary reporter genes include the chloramphenicol acetyltransferase gene (cat) from Tn9 of E. coli, the β-glucuronidase gene at the uidA locus of E. coli, green, red, or blue fluorescent protein genes, and the luciferase gene. Expression of the reporter gene is quantified at an appropriate time after introducing the DNA into the recipient cells.

[0070] General methods for constructing recombinant DNA capable of transforming target cells are well known to those of skill in the art, and similar compositions and construction methods can be utilized for producing DNA useful herein. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2002) provides suitable construction methods.

[0071] Recombinant DNA can be readily introduced into host cells, such as mammalian, yeast, or insect cells, by any process useful for introduction into a particular cell, e.g., transfection with an expression vector containing the recombinant DNA by physical or biological methods, resulting in a transformed (transgenic) cell harboring the recombinant DNA, and the DNA sequence of interest is expressed by the host cell. In one embodiment, at least one recombinant DNA introduced into a cell is maintained extrachromosomally. In one embodiment, at least one recombinant DNA is stably integrated into the genome of the host cell.

[0072] Physical methods for introducing recombinant DNA into host cells include calcium-mediated methods, lipofection, biolistics, microinjection, electroporation, etc. Biological methods for introducing DNA of interest into host cells include the use of DNA and RNA viral vectors. Viral vectors, such as retroviral or lentiviral vectors, have become a widely used method for inserting genes into eukaryotic, e.g., mammalian, e.g., human, cells. Other useful viral vectors for introducing genes into cells can be derived from poxviruses, e.g., vaccinia virus, herpes virus, adenovirus, adeno-associated virus, baculovirus, etc.

[0073] To confirm the presence of the recombinant DNA sequence in the host cell, various assays may be performed, including, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; biochemical assays, such as immunological methods (ELISAs and Western blots) or other molecular assays to detect the presence or absence of specific gene products.

[0074] RT-PCR can be performed to detect and quantify RNA produced from the introduced recombinant DNA fragment. This application of PCR first requires reverse transcription of the RNA into DNA using an enzyme, such as reverse transcriptase, followed by amplification of the DNA through the use of conventional PCR techniques. In most cases, PCR techniques are useful but will not verify the integrity of the RNA product. Further information about the nature of the RNA product can be obtained by Northern blotting. This technique verifies the presence of an RNA species and provides information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using Northern hybridization of dot or slot blots. These techniques are modifications of Northern blotting and only verify the presence or absence of an RNA species.

[0075] While Southern blotting and PCR can be used to detect the recombinant DNA fragment in question, they do not provide information about whether the recombinant DNA fragment is being expressed. Expression can be assessed by specifically identifying the peptide product of the introduced DNA sequence or by assessing phenotypic changes caused by expression of the introduced DNA fragment in the host cell.

[0076] The recombinant viruses described herein have modifications in their genomic sequences relative to the corresponding wild-type viral genome, i.e., the genome of the recombinant virus has modifications, including deletions, and optionally insertions, in regions associated with transcription and corresponding to sequences of nonstructural or nonglycosylated viral proteins. The mutations in the viral genome are effective to inhibit or prevent production of at least one functional viral protein from the genome when those sequences are present in a non-transgenic cell that supports viral replication. In one embodiment, the deletions comprise from one to up to several thousand nucleotides corresponding to coding regions for viral proteins, e.g., 1%, 10%, 50%, 90% or more of the sequence corresponding to coding regions for viral proteins. In one embodiment, the deleted sequence corresponds to a sequence having substantial homology, e.g., at least 80% or more, e.g., 85%, 90%, 95%, and up to 100%, or any integer in between, nucleic acid sequence homology, to the VP30 sequence and / or GP / sGP sequence. In one embodiment, the deletion comprises from one up to several hundred nucleotides, e.g., 1%, 10%, 50%, 90% or more, of sequence corresponding to at least the non-coding sequence between the NP and VP35 coding sequences. In one embodiment, the deleted sequence corresponds to a sequence with substantial homology, 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 homology to the non-coding sequence between the NP and VP35 coding sequences.

[0077] In one embodiment, the infectious viral genome of a replication-deficient single-stranded negative-strand RNA virus of the present invention contains a deletion in a transcription-related, nonstructural, or nonglycosylated protein in a sequence corresponding to that in the wild-type viral genome and contains a heterologous sequence that is nontoxic to host cells, including cells in the tissue to be immunized. In one embodiment, the heterologous sequence is a marker sequence, selection sequence, or other detectable or detectable sequence, such as GFP or luciferase, or a selectable gene, such as a drug resistance gene, e.g., the hygromycin B resistance gene or neomycin phosphotransferase, and the marker gene or selectable gene is not present in the host cell prior to introduction of the vector.

[0078] Pharmaceutical Composition For example, pharmaceutical compositions of the present invention, suitable for nasal, parenteral, or oral administration, e.g., intravenous, intramuscular, intranasal, topical, or subcutaneous administration, comprise one or more viral isolates, e.g., one or more recombinant, infectious, biologically encapsulated, single-stranded, negative-sense RNA viral isolates, and optionally further comprise sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions may further comprise adjuvants or excipients known in the art. The compositions are generally presented in the form of individual dosages (unit doses). Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, which may contain adjuvants or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, e.g., olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings may be used to increase skin permeability and facilitate antigen absorption. Liquid dosage forms for oral administration may generally comprise liposome solutions containing liquid dosage forms. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs, containing insoluble diluents commonly used by those skilled in the art, such as purified water. In addition to insoluble diluents, such compositions may also contain adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

[0079] When the composition is used for administration to an individual, it may further contain salts, buffers, adjuvants, or other substances that are desired to improve the effectiveness of the composition. In vaccines, adjuvants, substances that can enhance a specific immune response, may be used. Prior to presentation to the immune system, the adjuvant and the composition are normally mixed, or presented separately but within the same site of the tissue to be immunized.

[0080] In one embodiment, the pharmaceutical composition is part of a sustained release system, e.g., with a pump or formed into a polymeric compound (see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (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 sustained release systems are discussed in the review by Langer (Science, 249:1527 (1990)).

[0081] A pharmaceutical composition comprises a therapeutically effective amount of a virus and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency, such as a federal or state government or the like in the United States. A pharmacopoeia or other generally recognized pharmacopeia is intended for use in animals, and more specifically, 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 used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, water, ethanol, and the like. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. These compositions can also be formulated as suppositories. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. 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 an appropriate amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0082] The compositions may be administered systemically, e.g., orally, or intramuscularly, in combination with a pharmaceutically acceptable vehicle, e.g., an injectable diluent. For oral administration, the virus may be used in combination with one or more excipients in the form of ingestible capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions are believed to contain at least 0.1% of the active ingredient. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 and about 60% by weight of a given unit dosage form. The amount of active ingredient in such useful compositions is so great that an effective dosage level will be obtained.

[0083] The compositions may also contain: binders such as tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, alginic acid, or the like; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, oil of wintergreen, or cherry flavor. Various other ingredients may also be present. For example, a syrup or elixir may contain the virus, sucrose, or fructose as a sweetener, methyl and propylparabens as preservatives, dyes, and flavorings such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form, including sustained-release preparations or devices, is deemed pharmaceutically acceptable and substantially non-toxic in the amounts employed.

[0084] The composition can also be administered intravenously or intraperitoneally by infusion or injection. A solution of the virus can be prepared in water or a suitable buffer, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycols, 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.

[0085] Pharmaceutical dosage forms suitable for injection and infusion include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, optionally encapsulated in liposomes, suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the ultimate dosage form will be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic 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 undesired microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, such as sugars, buffers, or sodium chloride.

[0086] 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 filtered sterilization.

[0087] Useful liquid carriers include water, alcohols, or glycols, or water-alcohol / glycol binders, in which the virus is dissolved or dispersed at an effective concentration, optionally with the aid of a nontoxic surfactant. Adjuvants such as fragrances and additional antibacterial agents can be added to optimize the properties for a given use. The resulting liquid compositions can be applied from absorbent pads, impregnated bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0088] Useful dosages of the viruses of the invention can be defined by comparing their in vitro activity and in vivo activity in animal models.

[0089] Medicinal purposes Administration of a composition can be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the composition of the invention, which is a vaccine, is provided before any symptoms or clinical signs of infection with a pathogen appear. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. When provided prophylactically, the gene therapy composition of the invention is provided before any symptoms or clinical signs of a disease appear. The prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or clinical signs associated with a disease.

[0090] When provided therapeutically, viral vaccines are provided upon detection of symptoms or clinical signs of actual infection. Therapeutic administration of the compound contributes to attenuating any actual infection. When provided therapeutically, gene therapy compositions are provided upon detection of symptoms or clinical signs of the disease. Therapeutic administration of the compound contributes to attenuating symptoms or clinical signs of the disease.

[0091] Thus, the vaccine compositions of the invention can be provided either before the onset of infection (to prevent or attenuate an anticipated infection) or after the onset of actual infection. Similarly, for gene therapy, the compositions can be provided before the appearance of any symptoms or clinical signs of a disorder or disease, or after the observation of one or more symptoms.

[0092] A composition is considered to be "pharmacologically acceptable" if its administration can be used in a recipient mammal. Such an agent is considered 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, for example, if it amplifies at least one primary or secondary humoral or cellular immune response against at least one viral strain.

[0093] The "protection" provided need not be absolute, i.e., the influenza infection need not be totally protected against or eliminated, provided there is a statistically significant improvement compared to a control group or set of mammals. Protection may be limited to reducing the severity or rapidity of onset of symptoms or clinical signs of the viral infection.

[0094] Pharmaceutical administration The compositions of the invention can confer resistance to one or more pathogens, e.g., one or more viral, microbial, or parasitic strains, by either passive or active immunization. In active immunization, the live vaccine composition can be 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 collected and administered to a recipient suspected of having an infection caused by at least one viral strain.

[0095] Thus, the present invention includes methods for preventing or attenuating a disorder or disease, such as infection by at least one strain of a pathogen. As used herein, a vaccine is considered to prevent or attenuate a disease if its administration results in either a total or partial attenuation (i.e., suppression) of the clinical signs or symptoms of the disease, or results in total or partial immunity to an individual with the disease.

[0096] The at least one viral isolate of the present invention can be administered by any method that achieves its intended purpose. For example, administration of such compositions can be by various parenteral routes, such as subcutaneous, intradermal, intramuscular, intraperitoneal, intranasal, oral, or transdermal. Parenteral administration can be achieved by infusion or by gradual perfusion over time.

[0097] Exemplary regimens for preventing, suppressing, or treating a virus-associated condition are described herein and include administration of an effective amount of the vaccine composition, administered as a single treatment or repeated as boosting or additional doses, for example, over a period of one week to about 24 months, or any range or value therein.

[0098] In accordance with the present invention, an "effective amount" of a composition is one that is sufficient to produce the desired effect. It is understood that the effective amount may depend on the race, age, sex, health, and weight of the recipient, the type of concurrent treatment, if any, the number of treatments, and the effect desired. The ranges of effective amounts provided below are not intended to limit the invention and represent dosage ranges.

[0099] Exemplary dosages include, but are not limited to, about 10 4 From 10 8 FFU or PFU, 10 6 From 10 8 FFU or PFU, 10 6 From 10 10 FFU or PFU, or 10 8 From 10 12 FFU or PFU or more, or about 10 6 From 10 8 Number of particles, 10 8 From 10 10 Number of particles, or 10 10 From 10 12 In one embodiment, the dosage is about 10 4 From 10 8 FFU or PFU, 10 6 From 10 8 FFU or PFU, 10 6 From 10 10 FFU or PFU, or 10 8 From 10 10 It is either FFU or PFU.

[0100] Exemplary Adjuvants Adjuvants include, but are not limited to, aluminum, water-in-oil (W / O) emulsions, oil-in-water (O / W) emulsions, ISCOMs, liposomes, nano- or micro-particles, muramyl di- and / or tripeptides, saponins, non-ionic block copolymers, lipid A, cytokines, bacterial toxins, carbohydrates, and derivatized polysaccharides, and combinations of two or more of these adjuvants within an adjuvant system (AS).

[0101] Exemplary types of adjuvants include, but are not limited to, TLR3 antagonists, such as poly(I:C), TLR4 agonists, such as one or more components of microbial lipopolysaccharides, e.g., monophosphoryl lipid A (MPLA), MPL®, and synthetic derivatives, e.g., E6020, TLR5 agonists, such as microbial flagellins, TLR7 and 8 agonists, such as single-stranded RNA or imidazoquinolines (e.g., imiquimod, gardikimod, and R848), TLR9 agonists, such as CpG oligonucleotides and ISS immunostimulatory sequences and imidazoquinolines, NLRP3 inflammasome agonists, such as chitosan, and dual TLR1 / 2 agonists, e.g., Pam3CSK4, lipopeptides.

[0102] In one embodiment, the adjuvant comprises a natural product derived from tree bark which may be combined with a saponin, cholesterol or cholesterol-like molecule, such as squalene.

[0103] In one embodiment, the adjuvant comprises an oil-in-water (O / W) emulsion, for example, comprising MF59 or AS03 and optionally 2% squalene. In one embodiment, the adjuvant comprises two different adjuvants, for example, MPL and a saponin such as QS21, for example, within a liposome.

[0104] In one embodiment, the adjuvant comprises Incomplete Freund's Adjuvant (IFA), MF59®, GLA-SE, IC31®, CAF01, AS03, AS04, or ISA51, and may include alpha-tocopherol, squalene, and / or polysorbate 80 in an oil-in-water emulsion.

[0105] In one embodiment, the adjuvant comprises extracts and formulations prepared from Ayurvedic medicinal plants, including, but not limited to, Ashwagandha, Amla (Emblica officinalis), Panax notoginseng, Tinospora cordifolia, and Shatavari (Asparagus racemosus).

[0106] In one embodiment, the adjuvant comprises an aluminum salt, a saponin, a muramyl di- and / or tripeptide, Bordetella pertussis, and / or a cytokine.

[0107] In one embodiment, the adjuvant is not an alum or an aluminum salt.

[0108] In one embodiment, the adjuvant is mixed with the recombinant filovirus immediately prior to administration.

[0109] Exemplary Antigens Exemplary viral glycoproteins include, but are not limited to, those from an arenavirus such as Ebola virus, e.g., Zaire, Sudan, Bundibugyo, Tai Forest (formerly known as Côte d'Ivoire), or Reston, Marburg virus, Lassa virus; a bunyaviridae such as Crimean-Congo hemorrhagic fever virus or a hantavirus; or a flavivirus such as dengue, Zika, or yellow fever virus.

[0110] Exemplary parasite antigens include, but are not limited to, those from Plasmodium, Leishmania, Giardia, Cryptosporidium, or Cyclospore.

[0111] Exemplary microbial antigens include, but are not limited to, those from Vibrio, such as Vibrio cholerae, or Mycobacterium.

[0112] Example of recombinant filovirus preparation method Materials and Methods

[0113] Cells and Cell Lines. Vero cells (derived from African green monkey kidney) were grown in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), L-glutamine, vitamins, non-essential amino acid solution, and antibiotics. The VeroVP30 cell line was established by cotransfecting Vero cells with pCAG-VP30 (for VP30 expression) and pPur (Clontech, Mountain View, CA), a protein expression plasmid containing a puromycin resistance gene, using the TransIT LT-1 transfection reagent (Mirus, 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 using a polyclonal peptide antibody against VP30.

[0114] Human embryonic kidney 293T cells were grown in high-glucose Dulbecco's modified Eagle's medium containing 10% FCS, L-glutamine, and antibiotics. All cells were maintained at 37°C and 5% CO.

[0115] Flow cytometry. Cells were detached in phosphate-buffered saline (PBS) containing 0.02% EDTA and subsequently washed once with cold PBS supplemented with 2% FCS and 0.1% sodium azide (wash buffer). Cells were incubated with VP30 antibody for 20 minutes on ice. After washing with buffer, the cells were further incubated with VP30 antibody for 20 minutes on ice. After washing with buffer, the cells were further incubated with a secondary antibody labeled with fluorescein isothiocyanate (Zymed Laboratories, Carlsbad, CA). They were then washed with buffer and analyzed using a FACSCalibur and Cell Quest software (Becton Dickinson, Franklin Lakes, NJ).

[0116] Generation of EbolaΔVP30 virus. The pTM-T7G-Ebo-Rib plasmid, containing a full-length Ebola virus cDNA flanked by a T7 RNA polymerase promoter and ribozyme sequences, was described in Newmann et al. (2002). First, a fragment encompassing nucleotides 6180 to 10942 (representing the plus-strand antigenome) was subcloned into a kanamycin-resistant cloning vector. Next, the VP30 ORF was replaced with one encoding neo or eGFP, respectively, by a series of overlapping PCR amplification steps using Pfu Turbo (Stratagene, La Jolla, CA). The altered subgenomic fragment was then inserted back into the full-length Ebola virus cDNA plasmid using two unique restriction enzyme sites, SalI and SacI (Figure 1). The resulting plasmids, designated pTM-EbolaΔVP30-neo or -eGFP, were sequenced to confirm the replacement of the VP30 ORF and the absence of any undesired mutations.

[0117] Artificially generated Ebola virus was inoculated into 5 × 10 cells using a TransIT LT1 (Mirus, Madison, WI) in BSL-4 containment. 5293T cells were transfected with 1.0 μg of pTM-EbolaΔVP30 (Neumann et al., 2002). Five days after transfection, supernatants were collected, cell debris was removed by low-speed centrifugation, and virus was amplified in VeroVP30 cells at 37°C and 5% CO in growth medium containing 2% FCS in MEM supplemented with L-glutamine, vitamins, non-essential amino acid solution, and antibiotics without puromycin.

[0118] Plaque assay and immunostaining assay. To determine the titer of wild-type Ebola virus or Ebola ΔVP30 virus, 10-fold dilutions of the virus were adsorbed to confluent VeroVP30 or wild-type Vero cells for 1 hour at 37°C, after which any unbound virus was removed by washing the cells with growth medium. The cells were then covered with growth medium containing 1.5% methylcellulose (Sigma). Seven days postinfection, cells were fixed with 10% buffered formaldehyde solution, removed from BSL-4, permeabilized with 0.25% Triton X-100 in PBS for 10 minutes, and blocked for 60 minutes in 4% goat serum and 1% bovine serum albumin (BSA) in PBS. The cells were subsequently incubated with a 1:1000 dilution of mouse anti-VP40 monoclonal antibody for 60 minutes, washed with PBS, and incubated with a 1:1000 dilution of anti-mouse IgG-peroxidase-conjugated secondary antibody for 60 minutes (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). After washing with PBS, the cells were incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma) in PBS. The reaction was stopped by rinsing the cells with water.

[0119] Western blotting. Partially purified viruses were 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]), incubated at 100°C for 5 min, removed from BSL-4, and separated on a 4-20% polyacrylamide gel. Separated proteins were transferred to Western polyvinylidene difluoride membranes (Schleicher & Schuell, Sanford ME) and blocked overnight at 4°C with 5% skim milk in PBST (PBS with 0.05% Tween 20 [Sigma]). Blots were incubated with primary antibodies (mouse anti-NP, rabbit anti-VP35, rabbit anti-VP40, mouse anti-GP, rabbit anti-VP30, or mouse anti-VP24) for 60 min at room temperature, washed three times with PBST, incubated with the appropriate secondary antibodies, conjugated with horseradish peroxidase (Zymed) for 60 min, and finally washed three times with PBST. Blots were then incubated with Lumi-Light Western substrate (Roche, Indianapolis, IN) and exposed to X-ray film (Kodak, Rochester, NY).

[0120] RNA isolation and RT-PCR. Cell culture supernatant from virus-infected VeroVP30 cells was inactivated with guanidine thiocyanate buffer and removed from BSL-4. Viral RNA was isolated using an RNasey Mini kit (Qiagen, Valencia, CA). RT-PCR was performed using 1 μg of isolated RNA and Ebola virus-specific primers with a RobusT One-Step RT-PCR kit (Finnzyme, Espoo, Finland). The resulting PCR product was cloned into pT7Blue (Novagen, San Diego, CA) and sequenced.

[0121] Transmission electron microscopy. Ultrathin electron microscopy was performed as described by Noda et al. (2002). Briefly, at 36 h postinfection, VeroVP30 cells infected with EbolaΔVP30-neo virus were fixed and inactivated with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, removed from BSL-4, and postfixed in the same buffer with 2% osmium tetroxide. Cells were subsequently dehydrated through a graded series of ethanol followed by propylene oxide before embedding in Epon812 Resin mixture (TAAB Laboratories Equipment Ltd., Berkshire, UK). Thin sections were stained with 2% uranyl acetate and the Reynolds method and examined at 80 kV in a Hitachi H-7500 electron microscope.

[0122] Selection of escape mutants. Ebola ΔVP30-eGFP was diluted 10-fold (10 -1 from -6 ) and incubated with the indicated mAbs at concentrations of 250–500 μg mAb / mL for 60 minutes at 37°C. The virus / mAb mixture was inoculated onto VeroVP30 cells for 60 minutes. Virus was grown in the presence of antibody for 5 days. Subsequently, virus was harvested at the highest virus-positive dilution growing in the presence of mAbs (as defined by GFP expression) and passaged a total of 3–6 times in the presence of antibody. Viral RNA was isolated, amplified by RT-PCR, and its GFP sequence was determined by sequence analysis.

[0123] Production and Passaging of EbolaΔVP30-neo Virus. We previously generated a full-length cDNA clone, Zaire ebolavirus-Mayinga (Newmann et al., 2002). Using a series of overlapping PCR amplification steps, a subgenomic fragment encompassing nucleotides 6180 to 10942 of the viral genome (numbers refer to the plus-strand antigenome) was used to replace the VP30 ORF with that portion of neomycin (neo). After confirming the authenticity of the PCR fragment by sequence analysis, the altered subgenomic fragment was inserted into a full-length Ebola virus cDNA construct via unique SalI and SacI restriction enzyme sites (Figure 1), resulting in a deletion of the Ebola virus cDNA genome within the VP30 ORF. Artificial generation of Ebola virus from a plasmid was provided by flanking the viral DNA with a T7 RNA polymerase promoter and a hepatitis D virus ribozyme sequence (Neumann et al., 2002).

[0124] To amplify the VP30-deleted Ebola virus, a stable VeroE6 cell line (designated VeroVP30) was established by cotransfecting Vero cells with two proteins encoding VP30 (pCAG-VP30) and puromycin (pPur, Clontech), and cell clones resistant to 5.0 μg / mL puromycin were selected. VP30 expression in individual clones was determined by flow cytometry using an antibody against VP30. The clone with the highest percentage of cells expressing VP30 (>90% as measured by flow cytometry) was used for further studies to amplify the Ebola ΔVP30 virus.

[0125] Briefly, human embryonic kidney (293T) cells were transfected with a plasmid for transcription of VP30-deleted Ebola virus RNA, a plasmid for expression of Ebola virus NP, VP30, VP35, and L proteins, and a plasmid for expression of T7 RNA polymerase. Five days after transfection, VeroVP30 cells were incubated with undiluted supernatant from the transfected cells. Seven days later, the supernatant was collected, diluted 10-fold, and used to infect fresh VeroVP30 cells in the next passage. A total of seven passages were performed, using the highest dilution of inoculum that still produced replicating virus at each passage. The presence of replicating virus was assessed by cytopathic effect (CPE), and infected VeroVP30 cells were immunostained with an antibody to VP40. Supernatant from each passage was also incubated with wild-type Vero cells as a control. As expected, CPE and viral antigens were undetectable in wild-type Vero cells, demonstrating that replicating EbolaΔVP30-neo virus is restricted to VeroVP30 cells.

[0126] Because the appearance of CPE in infected VeroVP30 cells indicated the presence of infectious (but biologically contained) Ebola virus, further evidence was sought by examining the presence of viral particles in cell culture supernatants derived from infected VeroVP30 cells. Briefly, on day 5 after infection of VeroVP30 cells with EbolaΔVP30-neo virus, the supernatant was collected and partially purified with 20% sucrose. The precipitate was suspended in PBS and separated on a 4-20% polyacrylamide gel. Western blot analysis was performed using antibodies specific for the corresponding Ebola virus proteins. All viral proteins were detected (excluding L, for which antibodies were not available). The VP30 protein of the viral particles was confirmed to originate from VeroVP30 cells, while its associated protein was encoded by the EbolaΔVP30-neo virus. In contrast, no viral proteins were detected in control samples derived from wild-type Vero cells infected with EbolaΔVP30-neo virus.

[0127] Genetic Stability of Ebola ΔVP30-neo Virus. A major concern with the use of VP30-deleted Ebola viruses is potential recombination with the VP30 sequence integrated into the genome of the VeroVP30 helper cell line. Therefore, to assess the genomic stability of Ebola ΔVP30-neo virus, three independent passages (7 passages each) were performed. While Ebola ΔVP30-neo virus replicated in VeroVP30 cells, no viral replication was observed in wild-type Vero cells. Total viral RNA was isolated from the cell culture supernatant of infected VeroVP30 cells after 7 passages. 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 those of the Ebola ΔVP30 cDNA construct used for virus production. Thus, there was no evidence of recombination in any of the three independent passage studies, demonstrating the genetic stability of the Ebola ΔVP30-neo virus genome.

[0128] To further demonstrate the biosafety of Ebola ΔVP30-neo virus, Ebola ΔVP30-neo virus was collected after seven serial passages in Vero VP30 cells and used in three consecutive "blind" passages in wild-type Vero cells. Briefly, Vero cells were infected with Ebola ΔVP30-neo virus (passage 7) at a multiplicity of infection (moI) of 5. Six days later, the supernatant was used for the next "blind" passage and Western blot assay. No viral NP protein was detected after any "blind" passage (data not shown). After three consecutive "blind" passages, plaque assays and immunostaining were performed on wild-type Vero cells to confirm the absence of replicating Ebola virus. As expected, no replicating virus was detected. Together, these data further demonstrate the biosafety of the Ebola ΔVP30 system.

[0129] Growth Rate of Ebola ΔVP30-neo Virus. One of the major concerns raised by providing viral proteins in trans is that their amount, expression rate, or both may not match those in cells infected with wild-type virus, leading to reduced virus titers and / or abnormal virus particle morphology. To address this potential pitfall, we compared the growth rate of Ebola ΔVP30-neo with that of wild-type Ebola virus. VeroVP30 cells or wild-type Vero cells were infected with a high moI of 1.0 or a low moI of 0.01, and supernatants were collected every 24 hours. Ebola ΔVP30-neo virus titers were determined in VeroVP30 cells, while wild-type Ebola virus titers were determined in wild-type Vero cells. To determine virus titers, cells were overlaid with 1.5% methylcellulose, and after 7 days, VP40 expression was tested using an immunostaining assay. Ebola ΔVP30-neo virus replicated efficiently in VeroVP30 cells under both conditions tested, reaching 10 s by day 6 postinfection. 7Ebola ΔVP30-neo replication reached 100 focus-forming units (FFU) / mL. Replication of Ebola ΔVP30-neo was not detected in wild-type Vero cells; the low titers detected up to 3 days postinfection likely reflected input virus. Collectively, these findings demonstrate the biological containment of the Ebola ΔVP30 system. The replication rate of Ebola ΔVP30-neo in VeroVP30 cells was similar to that of wild-type Ebola virus in either VeroVP30 (Figure 3, upper panel, open circles) or wild-type Vero cells (Figure 3, lower panel, open circles), establishing the described approach as a highly efficient method for producing biologically contained Ebola virus.

[0130] Morphology of Ebola ΔVP30-neo virus. The morphology of Ebola ΔVP30-neo virus was next evaluated by transmission electron microscopy (TEM). VeroVP30 cells were infected with Ebola ΔVP30-neo virus and fixed 36 hours later. Samples were processed for TEM as described by Noda et al. (2002). As shown in Figure 4 (right panel), particles budding from VeroVP30 cells infected with Ebola ΔVP30-neo virus were indistinguishable in size and shape from wild-type Ebola virus. Therefore, providing VP30 protein in trans had no discernible effect on virus particle morphology, and the described system appears suitable for studying, for example, virus particle formation and budding.

[0131] Taken together, the above results demonstrate that Ebola ΔVP30-neo virus replicates to high titers in biocontained helper cell lines, is genetically stable, and is morphologically indistinguishable from wild-type virus particles. We provide proof-of-concept for the production of biocontained Ebola virus and evaluate the use of this strategy in basic research and drug screening applications.

[0132] Production of Ebola ΔVP30-eGFP virus and its utility for basic research applications. We produced an Ebola ΔVP30 virus encoding enhanced green fluorescent protein (eGFP) instead of VP30 (Figure 1; designated Ebola ΔVP30-eGFP) using the same steps described above for Ebola ΔVP30-neo virus. Similar to Ebola ΔVP30-neo virus, the eGFP variant produced 8.0 × 10 7 Virus replicated efficiently with titers reaching FFU / mL, and eGFP expression was observed as early as 10 hours postinfection (data not shown).

[0133] Takeda et al. (2003) used replication-competent vesicular stomatitis virus (VSV) pseudotyped with Ebola virus GP and two neutralizing monoclonal antibodies (mAbs) 133 / 3.16 and 226 / 8.1 to map Ebola virus GP epitopes and generate escape mutants. To identify authentic Ebola virus particles, Takeda et al. (2003) based their VSV-pseudotype system on the basis of which escape mutants were generated by amplifying Ebola ΔVP30-eGFP virus in the presence of mAb 133 / 3.16 or 226 / 8.1. Each of the eight escape mutants for mAb 133 / 3.16 had a histidine-to-arginine substitution at position 549 in the GP (H549R), as reported by Takada et al. (2003). Using mAb 266 / 8.1, we isolated 12 escape mutants, all of which contained an arginine-to-tryptophan substitution at position 134 (R134W), a mutation identical to that identified by Takada et al. (2003). However, none of the two escape mutants described by Takada et al. (2003) were detected. Whether this discrepancy in escape mutants reflects differences between the biological mechanisms used and random mutations is currently unknown. Nevertheless, these studies demonstrate one way in which biologically contained boraviridae viruses can be used in basic research applications.

[0134] Biocontained Ebola viruses lacking the VP30 gene provide a safe, alternative method for studying standard Ebola viruses, developing Ebola virus vaccines, and screening chemical libraries for compounds that interfere with the Ebola virus life cycle. Indeed, each of the three different biocontained viruses produced (encoding neomycin or eGFP instead of VP30) was biologically suppressed, but not wild-type, as demonstrated by their ability to replicate in VeroVP30 (a Vero cell line stably expressing VP30 in trans). Furthermore, viral titers were 10 7 FFU / mL and were therefore comparable to those obtained for wild-type Ebola virus (Figure 3; Volchov et al., 2001; Neumann et al., 2002; Ebihara et al., 2006), whereas morphological, biological, and virological analyses showed that the tested properties of Ebola ΔVP30 virus were indistinguishable from those of wild-type Ebola virus.

[0135] Exemplary Efficacy Protocol Ebola virus (Filoviridae) causes a severe hemorrhagic fever in humans and nonhuman primates with a fatality rate of up to 90% (Jonson et al., 1977). Currently, there are no licensed vaccines or antivirals available against Ebola virus. A vaccine against Ebola virus is desirable not only for the local population in endemic areas of Africa, but also for post-exposure treatment of healthcare workers during outbreaks and researchers after accidental exposure to the virus. A few vaccine candidates have demonstrated protection in mice, guinea pigs, or nonhuman primates against lethal Ebola virus challenge; however, each of these candidates has disadvantages, such as lack of protection in nonhuman primates, pre-existing immunity to the vector in humans, or potential central nervous system complications (Reed et al., 2007). Furthermore, current vaccine candidates are based on virus-like particles (VLPs) or viral vector vaccines, which do not express all components of the viral antigens. On the other hand, the use of live attenuated vaccines is not feasible for Ebola virus from a biosafety perspective. To overcome these potential limitations, biocontained viruses present an attractive option because they are biosafe but provide all viral antigens.

[0136] Materials and Methods Cells. VeroVP30 cells were established as described in Example 1 and grown in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), L-glutamine, vitamins, non-essential amino acid solution, and 5 μg / mL puromycin (Sigma, St. Louis, MO).

[0137] Virus. The Ebola ΔVP30 virus was produced as described in Example 1. Briefly, a plasmid containing the full-length Ebola cDNA genome of the Zaire Mayinga strain of Ebola virus was used to replace the VP30 open reading frame (ORF) with the ORF for the neomycin drug resistance gene. Using Ebola virus reverse genetics (Neumann et al., 2002), the Ebola ΔVP30 virus was produced and passaged in a Vero cell line stably expressing VP30 in MEM medium. Ebola ΔVP30 was propagated in Vero VP30 cells in MEM medium supplemented with 2% FCS as described above. The virus was harvested at a multiplicity of infection (MOI) of 1 6 days after infection of the cells and stored directly at -80°C. The harvested virus was also partially purified by ultracentrifugation in 20% sucrose at 27,000 rpm for 2 hours. The virus pellet was resuspended in sterile PBS and stored at -80°C. The recovered virus was also partially purified by ultracentrifugation with 20% sucrose at 27,000 rpm for 2 hours. The virus pellet was resuspended in sterile PBS and stored at -80°C. The virus titer was determined by plaque assay on confluent VeroVP30 cells overlaid with 2% FCS-MEM containing 1.5% methylcellulose (Sigma).

[0138] Because wild-type Ebola virus does not kill mice, a challenge study was performed with a mouse-adapted Ebola virus (Bray et al., 1998). This virus was produced as described in Ebihara et al., 2006.

[0139] Antibody titers. The concentration of Ebola glycoprotein (GP)-specific immunoglobulin G (IgG) antibodies in vaccinated mice was determined by 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 sera from control and vaccinated mice, bound antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) in an ELISA plate reader at a wavelength of 405 nm.

[0140] Intracellular staining and flow cytometry. Cytokine-producing CD8 + T cell numbers were determined by intracellular staining as described by Murali-Krishna et al. (1998). Briefly, splenocytes were treated with Ebola peptide NP. 279-288 (derived from the nucleoprotein NP, SFKAALSSLA; SEQ ID NO:16) ( Olinger et al., 2006 ; Simmons et al., 2004 ), VP40 171-180 (YFTFDLTALK, derived from matrix protein VP40; SEQ ID NO:17), or GP 161-169 The cells were stimulated with LYDRLASTV (derived from GP) (Olinger et al., 2005; Warfield et al., 2005) in the presence of brefeldin A and IL-2 for 5 h. Following activation, the cells were purified by using the Cytofix / Cytoperm kit from BD Biosciences (San Jose, CA). + and intracellular IFNγ. +The number of T cells was determined by using a FACSCalibur flow cytometer (BD Biosciences).

[0141] Vaccination and Challenge. Four-week-old female BALB / c mice (The Jackson Laboratory, Bar Harbor, ME) were anesthetized with isoflurane and given 10 mg of 10 ... 6 Mice were inoculated twice, 3 weeks apart, with 10 focus-forming units (FFU) of sucrose-purified Ebola ΔVP30 virus; control mice were inoculated simultaneously with PBS. A second group of mice received 100% PBS recovered from cell culture supernatant. 7 Mice were vaccinated three times (at 3-week intervals) with FFU virus or a control group was inoculated with 2% FCS-MEM. Vaccinations were performed at the University of Wisconsin-Madison. Mice were then transferred to a BSL-4 laboratory at the National Microbiology Laboratory of the Public Health Agency of Canada, where they were inoculated with a 50% lethal dose of mouse-adapted Ebola virus (MLD) per 1000 mice (Figure 1). 50 They were challenged with 100 mg of vaccinated IgG (i.e., the dose required to kill 50% of infected animals). On day 4 of challenge, virus titers were determined in the sera of three control and three vaccinated mice from each group. The remaining mice were monitored for survival for 28 days.

[0142] Antibody responses of mice immunized with Ebola ΔVP30 virus. To evaluate the Ebola ΔVP30 virus as a potential vaccine, its immunogenicity in mice was determined. Mice inoculated with Ebola ΔVP30 virus did not show any signs of disease, demonstrating the lack of pathogenicity of the Ebola ΔVP30 virus. To determine the concentration of antibodies to the Ebola glycoprotein (GP), serum samples were collected two weeks after each vaccination. When serum samples were tested for IgG antibodies by ELISA with purified GP, the inoculated animals showed elevated titers of antibodies to GP after the first inoculation compared with control mice, and these antibody titers further increased after the second and third inoculations. This finding demonstrates the ability of the biocontained Ebola ΔVP30 virus to elicit antibodies to GP.

[0143] CD8 in vaccinated mice + T cell response. Cellular responses to the vaccine in mice were tested. Mice were inoculated as described above. Eight days after the second vaccination, four inoculated and two control mice were euthanized and their spleens were removed. Splenocytes were isolated and injected with Ebola peptide NP. 279-288 (SFKAALSSLA), VP40 171-180 (YFTFDLTALK) or GP 161-169 The vaccinated mice were stimulated with Ebola peptide NP (LYDRLASTV) for 5 hours in the presence of brefeldin A and IL-2. 279-288 In cells stimulated with IFN-γ, the percentage of IFN-γ-positive CD8 + In control mice, IFNγ-positive CD8 + The number of cells was significantly lower, ranging from 0.00513% to 0.00794%. 171-180 or GP 161-169 IFNγ-positive CD8 stimulated by + No cells were detected (data not shown).

[0144] Protective efficacy of Ebola ΔVP30 virus in mice. To evaluate the protective efficacy of Ebola ΔVP30 virus, two groups of 4-week-old mice were inoculated intraperitoneally and subsequently lethally challenged with mouse-adapted Ebola virus. Mice in "Group 1" were 10 7 Eight control mice were inoculated in a similar manner with 2% FCS-MEM. Mice from this group were routinely killed using a 1000 MLD 50 Mice in "Group 2" were challenged with a mouse-adapted Ebola virus 7 weeks after the last immunization (Bray et al., 1998; Ebihara et al., 2006). 6 Mice from "Group 2" were inoculated twice (at 3-week intervals) with FFU of purified Ebola ΔVP30 virus; 10 control mice were inoculated simultaneously with PBS. 50 The mice were challenged 8 weeks after their last immunization with mouse-adapted Ebola virus. No signs of illness or disease were observed in mice inoculated with purified or unpurified Ebola ΔVP30 virus, while control mice from both groups began to show signs of illness (e.g., ruffled coat) along with weight loss 3 days after challenge. By day 7 post-challenge, all control mice had succumbed to infection. In contrast, vaccinated mice from both groups showed no signs of illness, as characterized by ruffled coat and weight loss, and were fully protected against lethal challenge for up to 28 days, at which time all surviving mice were euthanized. Four days post-challenge, mice were sacrificed, and virus titers in serum were determined. Vaccinated mice from both groups showed a 3-4 log increase in virus titer compared to their corresponding control mice. 10 Collectively, these data demonstrate that Ebola Δ30 virus effectively protects mice against challenge with a lethal dose of mouse-adapted Ebola virus. Similar results were obtained in guinea pigs.

[0145] Mice inoculated with EbolaΔVP30 were completely protected from a lethal challenge with mouse-adapted Ebola virus, and viral titers in serum from these mice were over 1000-fold lower than viral titers in control mice.

[0146] The humoral response to Ebola virus infection is important because it is demonstrated by protection from lethal challenge due to the active 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, classic vaccine approaches, such as γ-irradiated Ebola and Marburg viruses, along with GP expressed in baculovirus, have produced modest antibody responses; however, they have failed to protect mice against lethal challenge (Ignatyeve et al., 1996; Lupton et al., 1980; Mellquist-Riemenschneider et al., 2003). In contrast, Ebola and Marburg VPLs protect mice from lethal challenge with Ebola or Marburg viruses (Warifield et al., 2003, Warifield et al., 2004, Warifield et al., 2005) and induce not only humoral responses but also CD8 + Similarly, in non-human primates (NHPs), total protection from lethal challenge is mediated by humoral responses and CD8 + Vaccine candidates that protect NHPs from lethal Ebola virus challenge, such as recombinant vesicular stomatitis virus (VSV) (Jones et al., 2005) and adenovirus (Sullivan et al., 2000), have been shown to induce, to varying degrees, CD8+ responses in NHPs. +Induce T cell responses (Jones et al., 2005; Sullivan et al., 2000). + Whether T cell responses are sufficient to provide protection to NHPs from lethal Ebola virus infection remains to be determined.

[0147] Although vaccine candidates such as recombinant VSV or parainfluenza viruses offer protection in various animal models (Bukreyev et al., 2006; Jones et al., 2005), there are safety concerns regarding the use of these vaccines in humans (Bukreyev et al., 2006; Jones et al., 2005; Reed et al., 2007). Also, pre-existing immunity to recombinant adenovirus-based vaccines is limited by the high viral load (10 10 There is concern because of the high number of particles required 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 lethal challenge with these viruses (Warfield et al., 2004; Warfield et al., 2005). While VLPs are safe, and because of the rarity of Ebola virus infection, pre-existing immunity to Ebola or Marburg viruses is not a concern for VLP vaccines, which makes it difficult to produce larger quantities of VLPs from cell culture.

[0148] The biocontained Ebola ΔVP30 virus is therefore an ideal vaccine candidate because it combines the advantages of VLPs and vectored vaccines (i.e., it is safe and effective), but, like standard viruses, can be propagated to high titers in VeroVP30 cells (Example 1). Further studies will include testing the Ebola ΔVP30 virus for its protective efficacy in NHPs. In addition, shorter, single-vaccine protocols will be evaluated to determine if an Ebola ΔVP30 virus vaccine would induce rapid and effective immunity in the event of a pandemic or bioterrorist attack. This includes evaluating the Ebola ΔVP30 virus as a vaccine for post-exposure treatment.

[0149] The present invention will be further described in the following non-limiting examples. [Example]

[0150] Example 1 Nonhuman primates were vaccinated with one or two doses of vaccine virus (IM or aerosol) followed by a challenge (heterologous or homologous challenge) four weeks after the final dose. Some inoculations included one of three different adjuvants. Data from Study #2 demonstrate that this had a 10-fold higher vaccine virus yield than Study #1, which only included those animals vaccinated with virus and adjuvant that survived the heterologous challenge. In Study #3, in addition to the protocol used for the vaccine of the present invention (3A and 3D), two other anti-Ebola virus vaccines currently undergoing testing were tested (vaccine A, intranasal administration of a vaccine containing a replication-competent virus; vaccine B, intramuscular administration of a vaccine containing a replication-competent virus). In one protocol, inoculation alone induced vaccine A (3G). In other inoculation protocols, combinations of the vaccine of the present invention with vaccine A (3B and 3C) and combinations of vaccine A and vaccine B (3E and 3F) were tested. Some protocols included adjuvants (3A-3D). [Table 1] [Table 2]

[0151] Example 2 Exemplary Manufacturing Process Preparation of master virus seed (MVS)

[0152] Chemical transfection reagents for introducing plasmids for vaccine virus production by reverse genetic techniques are insufficient for VeroVP30 cells. Therefore, electroporation was performed using the Neon Transfection system. The Neon Transfection system was sterilized with ethylene oxide before use. Six microcentrifuge tubes, each containing 1 × 10e6 VeroVP30 cells, were mixed with a total amount of 10 μg of plasmid (pCAGGS EBOV L, pCAGGS EBOV NP-VP35, T7, and pTM EbolaΔVP30 plasmids in a 2:1:1:2 ratio) and electroporated with the Neon Transfection system using three 20-millisecond pulses at 1200 V. The transfected cells were seeded into each well of a 6-well plate and incubated at 37 ± 2°C and 5 ± 2% CO2 for 4 days before expansion into TC75 flasks.

[0153] Cell culture and vaccine virus recovery Cell cultures were initiated in complete medium (viral production-serum-free medium [VP-SFM, Thermo Fisher Scientific]) supplemented with 1% Glutamax and expanded into 18 x 10-layer cell factories (Nunc). The VP-SFM performed better than OptiVERO medium (InVitria), and the 10-layer cell factories performed better than hyperstacks (Corning) in terms of virus production, resulting in an increase in viral titer (1-1.5 log increase in titer expressed in focus-forming units).

[0154] When cells reached 80-90% confluency, each 10-layer cell factory was washed three times with DPBS and then infected with MVS at an MOI of 0.1 in VP-SFM with 1% Glutamax. Each infected 10-layer cell factory was incubated at 37±2°C and 5±2% CO2 for 7 days. Following the 7-day infection period, each 10-layer cell factory was tested for contamination and its contents were harvested into a sterile 20 L bioprocess bag. The pooled sample and its bulk harvest were pumped through a depth filter (1.2 μM filtration with a Sartorius2 XLG MidiCap) into a new sterile bioprocess bag.

[0155] Host genomic DNA was removed by benzonase treatment. MgCl2 was added to the filtered virus harvest to a final concentration of up to 2 mM MgCl2 and incubated for 4-6 hours at 37 ± 2°C. After benzonase treatment, β-propiolactone (BPL) was added to a final concentration of 0.1% v / v and incubated at 2-8°C for 16-18 hours.

[0156] Cleaved host DNA, Benzonase, BPL, and other host impurities were subsequently removed by tangential flow filtration (TFF). The Benzonase / BPL-treated virus harvest was concentrated approximately 10-fold (e.g., concentrated to avoid precipitation) and diluted with, e.g., Ca. 2+ / Mg 2+The resulting solution is dialyzed into Dulbecco's Phosphate Buffered Saline (DPBS) with 0.1 μm pore size, followed by further filtration using a sterilized / closed TFF system (the TFF filter is 0.1 μm pore size from GE Health, 30 cm patch length, e.g., GE RTPCFP-1E-8S hollow fiber cartridge) to obtain a ~2x concentration of the desired product (e.g., 6 x 10). 7 The TFF retentate and wash pool samples were concentrated to 1000 ng / mL (FFU / mL). After dialysis, samples of the TFF retentate and wash pool were dialyzed and titered by ELISA and host protein. Retentate and wash samples meeting host protein specifications (e.g., ≦500 ng / mL) were pooled, centrifuged to remove any remaining particulate material, transferred to a sterile 2 L Erlenmeyer flask, and stored at 2-8°C (e.g., in DPBS) until product manufacturing.

[0157] Testing of media and conditions When cells were cultured and infected using TC175 flasks, OPTIVERO medium resulted in virus titers approximately 1 log unit higher than VP-SFM (e.g., 6.84 log 10 FFU / mL [OPTIVero] vs. 5.75 log 10 FFU / mL [VP-SFM]). When cells are cultured and infected in a 10-tier system using VP-SFM medium, they produce titers that are approximately 1 log unit higher (e.g., 6.69 log units) than when cells are cultured and infected in a hyperstack using VP-SFM medium. 10 FFU / mL [10-stage system] vs. 5.58 log 10 FFU / mL [hyperstack]). VP-SFM medium contains plant hydrolysates (e.g., containing di- and tri-plant peptides) and ion chelators, while OPTIVERO medium contains recombinant human albumin, recombinant human transferrin (i.e., does not contain plant hydrolysates or ion chelators).

[0158] References Boehmann et al.,Virology,332:406(2005). Bray et al.,J.Infect.Dis.,178:651(1998). Bukreyev et al.,J.Virol.,80:2267(2006). Chan et al.,Cell,106:117(2001). Chandran et al.,Science,308:1643(2005). Ebihara et al.,Plos Pathogens,2:0705(2006). Ebihara et al.,PLos Pathog.,2:e73(2006). Feldmann et al.,in Virus Taxonomy:Eighth Report of the International Committee of Taxonomy of Viruses,eds.Fauquet,C.,Mayo,M.A.,Desselberger,U.,&Ball,L.A.(Elsevier,London),pp.645(2004). Groseth et al.,J.Virol.,79:4425(2005). Gupta et al.,J.Virol.,75:4649(2001). Halfmann et al.,In submission(2007). Hartman et al.,J.Virol.,80:6430(2006). Harty et al.,Mol.Cell,10:307(2002). Ignatyeve et al,J.Biotechnol.,44:111(1996). Jasenosky et al.,J.Virol.,75:5205(2001). Johnson et al.,Lancet,i:569(1977). Johnson et al.,Virol.J.,3:31(2006). Jones et al.,Nat.Med.,11:786(2005). Licata et al.,J.Virol.,78:7344(2004). Lupton et al.,Lancet,2:1294(1980). Manicassamy et al.,J.Virol.,79:4793(2005). Marzi et al., Virology, 352:345 (2006). McCarthy et al.,J.Virol.Methods,137:115(2006). Mellquist-Riemenschneider et al.,Virus Res.,92:187(2003). Modrof et al.,J.Biol.Chem.,277:33099(2002). Modrof et al., J.Virol., 77:3334 (2003). Muhlberger et al.,J Virol.,73;2333(1999). Murali-Krishna et al.,Immunity,8;177(1998). Neumann et al.,J.Virol.,76:406(2002). Neumann et al.,J.Virol.,79:10300(2005). Noda et al.,j.Virol.,76:4855(2002). Olinger et al.,J.Virol.,79:14189(2005). Panchal et al.,Proc.Natl.Acad.Sci.USA,100:15936(2003). Reed et al.,Vaccine.25:1923(2007). Sanchez et al.,in Fields Virology,eds.Knipe,DM,Howley,PM,Griffin,DE,Martin,MA,Lamb,RA,Roizman,B.,&Straus,SE(Lippincott,Williams&Wilkins,Philadelphia),pp.1409(2007). Sanchez et al.Virus Res.,29:215(1993). Shimojima et al.,J.Virol.,80:10109(2006). Simmons et al.,Virology,318:224(2004). Sullivan et al.,Nature,408:681(2000), Takada et al.,J.Virol.,75:2324(2001). Takada et al.,Proc,Natl.Acad.Sci.USA,94:14764(1997). Takada et al.,Vaccine,25:993(2007). Takada,J.Virol.,77:1069(2003). Theriault et al.,Arch.Virol.Suppl.,19:157(2005). Volchkov et al.,Science,291:1965(2001). Warfield et al.,J.Immunol.,175:1184(2005). Warfield et al.,Pro.Natl.Acad.Sci.USA,100:15889(2003). Warfield et al.,Vaccine,22:3495(2004). Wool-Lewis et al.,J.Virol.,72:155(1998). Yonezawa et al.,J.Virol.,79:918(2005).

[0159] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described, to some extent, with reference to preferred embodiments thereof, and numerous 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.

Claims

1. 1. A vaccine comprising an effective amount of a recombinant filovirus and one or more adjuvants, wherein the genome of the recombinant filovirus comprises a deletion of one or more nucleotides in the polynucleotide sequence of a viral protein corresponding to Ebola virus VP30, and wherein the deletion is effective to prevent expression of a functional viral protein corresponding to Ebola virus VP30 upon infection of a cell with the recombinant filovirus, the vaccine optionally comprising a pharmaceutically acceptable carrier, and the vaccine does not comprise alum or aluminum salts.

2. 2. The vaccine of claim 1, wherein at least 90% of the sequence corresponding to the VP30 sequence in the viral genome of the virus is deleted.

3. The vaccine of claim 1 or 2, wherein the genome further comprises a nucleotide sequence encoding a prophylactic or therapeutic heterologous gene product, and optionally, the nucleotide sequence is inserted within 1000 nucleotides of the deletion site or at the deletion site, the nucleotide sequence is inserted into the filovirus genome at a site other than the deletion site within the polynucleotide, the nucleotide sequence replaces a GP / sGP sequence or part thereof, or the nucleotide sequence is inserted within the GP / sGP coding sequence.

4. 4. The vaccine of claim 3, wherein the nucleotide sequence is inserted between the NP coding sequence and the VP35 coding sequence within the filovirus genome.

5. 5. The vaccine of claim 3 or 4, wherein the heterologous gene product comprises a glycoprotein or a non-glycoprotein, and the glycoprotein or non-glycoprotein is optionally a Marburg virus, Ebola virus, Sudan virus, Tai Forest virus, Reston virus, or Bundibugyo virus glycoprotein, Ebola NP, Ebola VP40, Ebola VP35, Marburg NP, Marburg NP VP40, Marburg NP VP35, Plasmodium circumsporozoite protein (CSP), Plasmodium apical membrane protein (AMA), Plasmodium rhoptry neck protein 2 (RON2), Plasmodium RH5, flavivirus membrane protein, flavivirus envelope protein, or Bunyaviral glycoprotein precursor (GPC) protein.

6. The vaccine of any one of claims 1 to 5, wherein the recombinant filovirus genome is a recombinant Ebola virus genome.

7. The vaccine of any one of claims 1 to 6, wherein the adjuvant comprises lipopolysaccharide, squalene, or saponin.

8. 8. The vaccine of claim 7, wherein the lipopolysaccharide comprises monophosphoryl lipid A or the saponin comprises an extract of Quillaja japonica.

9. A composition for use in the manufacture of a medicament for conferring immunity to a mammal, comprising an effective amount of a vaccine according to any one of claims 1 to 8.

10. A recombinant filovirus, wherein the genome of the recombinant filovirus comprises a first deletion comprising one or more nucleotides in a polynucleotide sequence of a viral protein corresponding to Ebola virus VP30, the first deletion being effective to prevent expression of a functional viral protein corresponding to Ebola virus VP30 upon infection of a cell with the recombinant filovirus, and the genome comprises a mutation in a region flanked by the NP coding sequence and the VP35 coding sequence, a mutation in the GP / sGP coding sequence, and / or an insertion within 1000 nucleotides of or at the site of the first deletion, or a combination thereof, wherein the genome comprises one or more filoviruses. a vaccine encoding a GP / sGP glycoprotein, wherein the mutation in the region flanking the NP and VP35 coding sequences comprises an insertion of a nucleotide sequence encoding a prophylactic or therapeutic heterologous gene product and, optionally, a deletion of one or more nucleotides in the region flanking the NP and VP35 coding sequences, wherein the mutation in the GP / sGP coding sequence comprises an insertion of a nucleotide sequence encoding a prophylactic or therapeutic heterologous gene product and, optionally, a deletion of one or more nucleotides within the GP / sGP coding sequence, or wherein the insertion within 1000 nucleotides of or at the site of the first deletion encodes a prophylactic or therapeutic gene product.

11. 11. The vaccine of claim 10, which has a mutation in the region adjacent to the NP coding sequence and the VP35 coding sequence, or a mutation in the GP / sGP coding sequence, or an insertion encoding a prophylactic or therapeutic heterologous gene product adjacent to or in the deletion of the VP30 sequence.

12. 10. The vaccine of claim 1, which is a multivalent vaccine.

13. 1. A method for producing a recombinant filovirus, comprising: providing a supernatant from mammalian helper cells expressing a recombinant filovirus genome cultured in serum-free medium to produce progeny filovirus, wherein the genome of said recombinant filovirus comprises a deletion of one or more nucleotides in a polynucleotide sequence of a viral protein corresponding to Ebola virus VP30, and wherein said deletion is effective to prevent expression of a functional viral protein corresponding to Ebola virus VP30 upon infection of cells with the recombinant filovirus, and wherein said mammalian helper cells express a viral protein corresponding to Ebola virus VP30; contacting the supernatant with DNase and a virus inactivating agent, thereby providing an inactivated virus preparation; and A method comprising concentrating an inactivated virus preparation.

14. The method of claim 13, wherein the cells are Vero cells.

15. 15. The method of claim 13 or 14, wherein the collected supernatant is filtered before contacting with the DNase or viral inactivating agent, and optionally filtering the collected supernatant through a 0.5 to 5 micron filter or a 1 to 5 micron filter.

16. 16. The method of any one of claims 13 to 15, wherein the inactivated virus preparation is subjected to filtration, and optionally, said filtration of the inactivated virus preparation is through a 0.01 to 1 micron filter or a 0.05 to 0.25 micron filter.

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