Patent 6,649,372

The present invention relates methods of generating infectious negative-strand virus in host cells by an entirely vector-based system without the aid of a helper virus. In particular, the present invention relates methods of generating infectious recombinant negative-strand RNA viruses intracellularly in the absence of helper virus from expression vectors comprising cDNAs encoding the viral proteins necessary to form ribonucleoprotein complexes (RNPs) and expression vectors comprising cDNA for genomic viral RNA(s) (vRNAs) or the corresponding cRNA(s). The present invention also relates to methods of generating infectious recombinant negative-strand RNA viruses which have mutations in viral genes and/or which express, package and/or present peptides or polypeptides encoded by heterologous nucleic acid sequences. The present invention further relates the use of the recombinant negative-strand RNA viruses or chimeric negative-strand RNA viruses of the invention in vaccine formulations and pharmaceutical compositions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of generating infectious negative-strand RNA viruses intracellularly from recombinant nucleic acid molecules. In particular, the present invention provides methods of generating an infectious negative-strand RNA virus in 293T cells, said methods comprising providing expression vectors capable of expressing genomic or antigenomic viral RNA segments,and nucleoproteins, and RNA dependent RNA polynerase, whereby RNPs are formed in said cells and infectious recombinant negative-strand RNA is produced in the absence of helper virus. The present invention encompasses methods of infectious recombinant negative-strand RNA virus having a segmented or non-segmented genome.

The present invention provides methods of generating infectious, replicating recombinant negative-strand RNA virus in the absence of helper virus by transiently transfecting 293T cells with expression vectors providing the genomic vRNA(s) or the corresponding cRNA(s) and the required viral proteins. In one embodiment, an infectious, replicating negative-strand RNA virus is generated in 293T cells by a method comprising: (a) introducing expression vectors which direct the expression of each required genomic vRNA segment or the corresponding cRNA into said cells; (b) introducing expression vectors which express a nucleoprotein and RNA-dependent RNA polymerase subunits or one or more additional viral proteins in said cells; and (c) culturing said cells such that RNPs are formed and the infectious, replicating recombinant negative-strand RNA virus is produced in the absence of helper virus. In accordance with these embodiments, each set of expression vectors may each comprise one or more vectors and each set of expression vectors may be introduced by transfection methods described herein or known to those of skill in the art.

The present invention also provides methods of generating infectious, replicating recombinant negative-strand RNA virus in the absence of helper virus by transfecting 293T cell lines expressing one or more genomic vRNAs or the corresponding cRNAs with expression vectors directing the expression of the required viral proteins. In a specific embodiment, an infectious, replicating recombinant negative-strand RNA virus is generated in a 293T cell line expressing genomic vRNA(s) or the corresponding cRNA(s) by a method comprising: (a) introducing expression vectors which express in said cells a nucleoprotein and RNA-dependent RNA polymerase subunits; and (b) culturing said cells such that RNPs are formed and the infectious, replicating virus is produced in the absence of helper virus.

The present invention also provides methods of generating infectious, replicating recombinant negative-strand RNA virus in the absence of helper virus in a 293T cell line that expresses one or more viral proteins required to form RNPs (i.e., nucleoprotein and RNA-dependent RNA polymerase subunits), said methods comprising: (a) introducing one or more expression vectors directing the expression of genomic vRNA(s) or the corresponding cRNA(s) in said cell line; (b) introducing one or more expression vectors that direct the expression of any viral proteins required to form RNPs which are not expressed by the 293T cell line; and (c) culturing said cell lines such that RNPs are formed and the infectious recombinant virus is produced. In accordance with this embodiment, each set of expression vectors may each comprise one or more vectors. For example, in the generation of an infectious, replicating negative-strand RNA virus with a nonsegmented genome, the first set of expression vectors would comprise one expression vector.

In accordance with the present invention, the 293 T cells, or any other host cell used in the methods of the invention, may be modified in many ways in order to facilitate rescue of a recombinant negative strand RNA virus in the absence of helper virus. In particular, the host cell may be modified or engineered to express viral proteins required for replication or packaging, either constitutively or inducibly. In either event, expression of the viral proteins is regulated by either a constitutive or inducible promoter as described herein or known to those of skill in the art. In such an embodiment, the host cell may be engineered to express viral proteins required to form RNPs or viral structured proteins. In another embodiment, the host cell may be modified to constitutively or inducibly expresses RNA-dependent RNA polymerases, or subunits thereof.

The present invention also provides methods of generating infectious, non-replicating or attenuated negative-strand RNA virus in 293T cells in the absence of helper virus, wherein method comprises introducing expression vectors which do not encode all of the genomic viral sequences required to form viral particles; or introducing expression vectors which provide the genomic vRNA(s) or corresponding cRNA(s) which contain a mutation, deletion or insertion which result in a recombinant virus with an attenuated phenotype. Further, the expression vectors may be introduced by transfection methods described herein or known to those of skill in the art.

The present invention also provides methods of generating infectious negative strand RNA virus in 293T cells infected by a helper virus, said methods comprising: (a) introducing expression vectors directing the expression of one or more vRNAs or the corresponding cRNAs in said cells; (b) introducing expression vectors directing the expression of one or more viral proteins in said cells; and (c) culturing the cells such that the RNPs are formed and the infectious, replicating negative-strand RNA virus is produced. In one embodiment, the helper virus provides viral proteins required to form the RNPs. In a preferred embodiment, the helper virus provides a DNA-dependent RNA polymerase such as, for example, bacteriophage T7, T3 or the SP6 polymerase. Preferably, the helper virus is not a negative-strand RNA virus and more preferably the helper virus is a DNA virus such as vaccinia.

The present invention also provides methods of generating infectious negative strand RNA virus in a 293T cell line infected by helper virus by introducing one or more expression vectors into said cell line. Accordingly, the 293T cell lines are transfected with expression vectors that direct the expression of vRNA(s) or the corresponding cRNA(s) and expression vectors that direct the expression of the viral proteins required for the formation of RNPs which are not provided by the helper virus.

The present invention also provides methods of generating an infectious recombinant negative-strand RNA viruses having greater than 3 genomic vRNA segments in mammalian cells, said methods comprising: (a) expressing genomic vRNA segments or the corresponding cRNAs from a first set of expression vectors in said cells; and (b) expressing a nucleoprotein and an RNA-dependent RNA polymerase from a second set of recombinant expression vectors in said cells, whereby ribonucleoprotein complexes are formed and the infectious recombinant negative-strand RNA viruses are produced in the absence of helper virus. Preferably, the infectious recombinant negative-strand RNA virus is a member of the Orthomyxoviridae family and most preferably the infectious recombinant negative-strand RNA virus is an influenza virus.

The present invention encompasses the generation of infectious recombinant negative-strand RNA viruses having greater than 3 genomic segments which are capable of replicating and producing progeny. The invention also encompasses the infectious recombinant negative-strand RNA viruses having greater than 3 genomic segments which are not capable of replicating and producing progeny.

In one embodiment, an infectious recombinant negative-strand RNA virus having greater than 3 genomic vRNA segments is generated in mammalian cells by a method comprising: (a) introducing a first set of expression vectors capable of expressing in said cells genomic vRNA segments or the corresponding cRNAs; (b) introducing a second set of expression vectors capable of expressing in said cells a nucleoprotein and RNA-dependent RNA polynerase; and (c) culturing said cells such that RNPs are formed and the infectious recombinant negative-strand RNA virus is produced in the absence of helper virus.

In another embodiment, an infectious recombinant negative-strand RNA virus having greater than 3 genomic vRNA segments is generated in a mammalian cell line expressing a nucleoprotein and an RNA-dependent RNA polynerase by a method comprising: (a) introducing expression vectors capable of expressing geniomic vRNA segments or the corresponding cRNAs; and (b) culturing said cells such that RNPs are formed and the infectious recombinant negative-strand RNA virus is produced in the absence helper virus. In another embodiment, an infectious recombinant negative-strand RNA virus having greater than 3 genomic vRNA segments is generated in a mammalian cell line expressing genomic vRNA segments or the corresponding cRNAs by a method comprising: (a) introducing expression vectors capable of expressing a nucleoprotein and an RNA-dependent RNA polymerase; and (b) culturing said cells such that RNPs are formed and the infectious recombinant negative-strand RNA virus is produced in the absence of helper virus.

The present invention also provides methods of generating infectious recombinant negative-strand RNA viruses having greater than 3 vRNA segments in the presence of helper virus by introducing into host cells expression vectors. The expression vectors introduced into the host cells comprise vectors directing the expression of greater than 3 vRNA segments or the corresponding cRNAs. Further, the expression vectors introduced into the host cells may comprise cDNA encoding one or more viral proteins, particularly one or more viral proteins required to form the RNPs.

The present invention provides methods of generating an infectious, replicating recombinant Newcastle disease virus (NDV) in mammalian cells, said methods comprising: (a) expressing genomic vRNA or the corresponding cRNA from an expression vector in said cells; and (b) expressing a nucleoprotein and an RNA-dependent RNA polymerase from a set of expression vectors in said cells, whereby ribonucleoprotein complexes are formed and the recombinant NDV is produced in the absence of helper virus. The present invention provides methods of generating an infectious, non-replicating recombinant Newcastle disease virus (NDV) in mammalian cells, said methods comprising: (a) expressing a vRNA or the corresponding cRNA from an expression vector in said cells, wherein said vRNA or the corresponding cRNA do not encode of the genomic viral proteins necessary for replicating; and (b) expressing a nucleoprotein and an RNA-dependent RNA polymerase from a set of expression vectors in said cells, whereby ribonucleoprotein complexes are formed and the non-replicating recombinant NDV is produced in the absence of helper virus.

In one embodiment, an infectious recombinant NDV is generated in mammalian cells by a method comprising: (a) introducing an expression vectors capable of expressing in said cells a genomic vRNA or the corresponding cRNA; (b) introducing a set of expression vectors capable of expressing in said cells a nucleoprotein and RNA-dependent RNA polynerase; and (c) culturing said cells such that RNPs are formed and recombinant NDV is produced in the absence of helper virus.

In another embodiment, an infectious recombinant NDV is generated in a host cell line expressing a nucleoprotein and an RNA-dependent RNA polymerase by a method comprising: (a) introducing expression vectors capable of expressing in said cell line a genomic vRNA segment or the corresponding cRNA; and (b) culturing said cell line such that RNPs are formed and recombinant NDV is produced in the absence helper virus. In another embodiment, an infectious recombinant NDV is generated in a host cell line expressing a genomic vRNA or the corresponding cRNA by a method comprising: (a) introducing expression vectors capable of expressing in said cell line a nucleoprotein and an RNTA-dependent RNA polymerase; and (b) culturing said cell line such that RNPs are formed and recombinant NDV is produced in the absence of helper virus.

The present invention also encompasses methods of generating NDV in the presence of helper virus by introducing expression vectors. The expression vectors directing the expression of genomic vRNA or cRNA and/or one or more viral proteins.

The ability to reconstitute negative-strand RNA viruses intracellularly in mammalian cells allows for the design of recombinant viruses (i.e., chimeric viruses) which express heterologous nucleic acid sequences or mutant viral genes. The heterologous sequences may encode, for example, epitopes or antigens of pathogens or tumors. The ability to reconstitute negative-strand RNA viruses intracellularly also allows the design of novel recombinant viruses (i.e., chimeric viruses) which express genes from different strains of viruses. Thus, the present invention provides methods of generating chimeric viruses which express heterologous nucleic acid sequences, mutant viral genes, or viral genes from different strains of virus intracellularly from expression vectors in the absence or presence of helper virus.

The present invention encompasses the cells and cell lines produced in the process of generating infectious negative-strand RNA viruses.

The infectiousness of a recombinant or chimeric negative-strand RNA virus of the present invention will vary depending upon the strain of virus from which the nucleic acid sequences encoding structural proteins such as influenza virus HA or NA are derived. Additionally, the infectiousness of a recombinant or chimeric negative-strand RNA virus of the invention will vary depending upon whether or not mutations have been introduced into the nucleic acid sequences encoding structural proteins. For example, a recombinant influenza virus of the invention with a mutation in HA may not be as infectious as another recombinant influenza virus expressing identical viral proteins without a mutation in HA.

The infectious recombinant or chimeric viruses of the present invention may or may not be capable of replicating and producing progeny. In a specific embodiment, an infectious recombinant negative-strand RNA virus of the invention is capable of replicating and producing progeny. The replication of an infectious recombinant or chimeric negative-strand RNA virus of the invention will vary depending upon the strain of virus from which the genomic vRNA(s) or the corresponding cRNA(s) were derived. Further, the replication of an infectious recombinant or chimeric negative-strand RNA virus of the invention will vary depending upon whether or not mutations have been introduced into the genomic vRNA(s) or the corresponding cRNA(s). For example, an infectious recombinant influenza virus expressing a truncated NS1 protein may replicate better than an infectious recombinant influenza virus expressing identical viral proteins except that it expresses a full-length NS1 protein.

The present invention provides for the use of the recombinant negative-strand RNA viruses or chimeric viruses of the invention to formulate vaccines against a broad range of viruses and/or antigens including tumor antigens. The recombinant negative-strand RNA viruses or chimeric viruses of the present invention may be used to modulate a subject's immune system by stimulating a humoral immune response, a cellular immune response or by stimulating tolerance to an antigen. When delivering, tumor antigens, the invention may be used to treat subjects having a disease amenable to immunity mediated rejection, such as non-solid tumors or solid tumors of small size. It is also contemplated that delivery of tumor antigens by the recombinant negative-strand RNA viruses or chimeric viruses described herein will be useful for treatment subsequent to removal of large solid tumors. The recombinant negative-strand RNA viruses or chimeric viruses of the invention may also be used to treat subjects who are suspected of having cancer.

The present invention also provides for the use of the recombinant negative-strand RNA viruses or chimeric viruses of the invention in pharmaceutical compositions for the administration of one or more peptides or polypeptides of interest.

5.1. EXPRESSION VECTORS FOR VRNA

Expression vectors comprising cDNA for viral RNA(s) or corresponding cRNA(s) will preferably be under the control of a DNA-dependent RNA polymerase promoter sequence. Examples of DNA-dependent RNA polymerase promoters include but are not limited to, bacterial promoters, viral promoters such as T7, T3 or SP3, and cellular promoters such as a mammalian RNA polymerase I promoter. Preferably, the cDNA for the viral RNA(s) or corresponding cRNA(s) is derived from a mammalian RNA polymerase I (RNA Pol I) promoter. Particularly preferred for this purpose is the truncated human RNA Pol I promoter consisting of nucleotides -250 to -1 of the corresponding native promoter or a functional derivative thereof (Jones et al., 1988, Proc. Natl. Acad. Sci. USA 85:669-673). In yet another embodiment, the vRNA(s) or corresponding cRNA(s) may be under the control of a mammalian RNA polymerase II promoter or RNA polymerase III promoter (see, e.g. Legin in Genes, Oxford University Press, New York (1977), pp. 819-22). To ensure the correct 3' end of each expressed vRNA or cRNA, each vRNA or cRNA expression vector will incorporate a ribozyme sequence or appropriate terminator sequence downstream of the RNA coding sequence. This may be, for example, the hepatitis delta virus genomic ribozyme sequence or a functional derivative thereof, or the murine rDNA terminator sequence (Genbank Accession Number M12074). Alternatively, for example, a Pol I terminator may be employed (Neumann et al., 1994, Virology 202:477-479). The RNA expression vectors may be constructed in the same manner as the vRNA expression vectors described in Pleschka et al., 1996, J. Virol. 70:4188-4192.

In a specific embodiment of the present invention, vRNA or cRNA expression vectors for the production of infectious recombinant NDV comprise the nucleotide sequence of the 3' termini of the NDV negative-sense genome RNA first identified by the Applicants'. This 3' termini of the NDV negative-sense genome RNA differs significantly from the NDV 3' termini sequence previously disclosed by Collins et al. in Fundamental Virology 3rd Ed. 1996 by Lippincott-Raven Publishers as shown in FIG. 6. The identification of the correct nucleotide sequence of the NDV 3' termini allows for the first time the engineering of recombinant NDV RNA templates, the expression of the recombinant RNA templates and the rescue of recombinant NDV particles.

A DNA-dependent RNA polymerase which recognizes the promoter sequence in the vRNA or corresponding cRNA expression vectors is used to produce the vRNA or corresponding cRNA from the nucleic acid sequences. Examples of DNA-dependent RNA polymerases include, but are not limited to, viral DNA-dependent RNA polymerase such as T7, T3 or the SP6 polymerase, bacterial DNA-dependent RNA polymerase, and cellular DNA-dependent RNA such as mammalian RNA polymerase I. In one embodiment, the expression vectors comprising the cDNA directing the expression of vRNA(s) or corresponding cRNA(s) are introduced into a host cell that does not express the DNA-dependent RNA polymerase which recognizes the DNA-dependent RNA polymerase promoter and one or more vectors expressing the DNA-dependent RNA polymerase subunits are introduced into said host cell. In accordance with this embodiment, the vectors expressing the DNA-dependent RNA polymerase subunits may be regulated by an inducible promoter. The expression of the DNA-dependent RNA polynierase then regulates the expression of the vRNA(s) or corresponding cRNAs.

The present invention provides expression vectors directing the expressing of genomic vRNA(s) or corresponding cRNA(s) which have one or more mutations. These mutations may result in the attenuation of the virus. For example, the vRNA segments may be the vRNA segments of an influenza A virus having an attenuated base pair substitution in a pan-handle duplex promoter region, in particular, for example, the known attenuating base pair substitution of A for C and U for G at position 11-12' in the duplex region of the NA-specific vRNA (Fodor et al., 1998, J. Virol. 6923-6290). By using the methods of the invention to produce recombinant negative-strand RNA virus, new attenuating mutations may be identified.

Sequences heterologous to a viral genome may be engineered into expression vectors directing the expression of vRNA(s) or corresponding cRNA(s) and introduced into host cells along with expression vectors directing the expression of viral proteins to generate novel infectious recombinant negative-strand RNA viruses or chimeric viruses. Heterologous sequences which may be engineered into these viruses include antisense nucleic acids and nucleic acid such as a ribozyme. Alternatively, heterologous sequences which express a peptide or polypeptide may be engineered into these viruses. Heterologous sequences encoding the following peptides or polypeptides may be engineered into these viruses include: 1) antigens that are characteristic of a pathogen; 2) antigens that are characteristic of autoimmune disease; 3) antigens that are characteristic of an allergen; and 4) antigens that are characteristic of a tumor. For example, heterologous gene sequences that can be engineered into the chimeric viruses of the invention include, but are not limited to, epitopes of human immunodeficiency virus (HIV) such as gp160; hepatitis B virus surface antigen (HBsAg); the glycoproteins of herpes virus (e.g., gD, gE); VP1 of poliovirus; and antigenic determinants of nonviral pathogens such as bacteria and parasites to name but a few.

Antigens that are characteristic of autoimmune disease typically will be derived from the cell surface, cytoplasm, nucleus, mitochondria and the like of mammalian tissues, including antigens characteristic of diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, pernicious anemia, Addison's disease, scleroderma, autoimmune atrophic gastritis, juvenile diabetes, and discoid lupus erythromatosus.

Antigens that are allergens are generally proteins or glycoproteins, including antigenis derived from pollens, dust, molds, spores, dander, insects and foods.

Antigens that are characteristic of tumor antigens typically will be derived from the cell surface, cytoplasm, nucleus, organelles and the like of cells of tumor tissue. Examples include antigens characteristic of tumor proteins, including proteins encoded by mutated oncogenes; viral proteins associated with tumors; and glycoproteins. Tumors include, but are not limited to, those derived from the types of cancer: lip, nasopharynx, pharynx and oral cavity, esophagus, stomach, colon, rectum, liver, gall bladder, pancreas, larynx, lung and bronchus, melanoma of skin, breast, cervix, uterine, ovary, bladder, kidney, uterus, brain and other parts of the nervous system, thyroid, prostate, testes, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and leukemia.

In one specific embodiment of the invention, the heterologous sequences are derived from the genome of human immunodeficiency virus (HIV), preferably human immunodeficiency virus-1 or human immunodeficiency virus-2. In another embodiment of the invention, the heterologous coding sequences may be inserted within an negative-strand RNA virus gene coding sequence such that a chimeric gene product is expressed which contains the heterologous peptide sequence within the viral protein. In such an embodiment of the invention, the heterologous sequences may also be derived from the genome of a human immunodeficiency virus, preferably of human immunodeficiency virus-1 or human immunodeficiency virus-2.

In instances whereby the heterologous sequences are HIV-derived, such sequences may include, but are not limited to sequences derived from the env gene (i.e., sequences encoding all or part of gp160, gp120, and/or gp41), the pol gene (i.e., sequences encoding all or part of reverse transcriptase, endonuclease, protease, and/or integrase), the gag gene (i.e., sequences encoding all or part of p7, p6, p55, p17/18, p24/25) tat, rev, nef, vif, vpu, vpr, and/or vpx.

One approach for constructing these hybrid molecules is to insert the heterologous coding sequence into a DNA complement of a negative-strand RNA virus gene so that the heterologous sequence is flanked by the viral sequences required for viral polymerase activity; i.e., the viral polymerase binding site/promoter, hereinafter referred to as the viral polymerase binding site, and a polyadenylation site. In an alternative approach, oligonucleotides encoding the viral polymerase binding site, e.g., the complement of the 3'-terminus or both termini of the virus genomic segments can be ligated to the heterologous coding sequence to construct the hybrid molecule. The placement of a foreign gene or segment of a foreign gene within a target sequence was formerly dictated by the presence of appropriate restriction enzyme sites within the target sequence. However, recent advances in molecular biology have lessened this problem greatly. Restriction enzyme sites can readily be placed anywhere within a target sequence through the use of site-directed mutagenesis (e.g., see, for example, the techniques described by Kunkel, 1985, Proc. Natl. Acad. Sci. U.S.A. 82:488). Variations in polymerase chain reaction (PCR) technology, described, also allow for the specific insertion of sequences (i.e., restriction enzyme sites) and allow for the facile construction of hybrid molecules. Alternatively, PCR reactions could be used to prepare recombinant templates without the need of cloning. For example, PCR reactions could be used to prepare double-stranded DNA molecules containing a DNA-directed RNA polymerase promoter (e.g., bacteriophase T3, T7 or SP6) and the hybrid sequence containing the heterologous gene and the polymerase binding site. RNA templates could then be transcribed directly from this recombinant DNA. In yet another embodiment, the recombinant vRNAs or corresponding cRNAs may be prepared by ligating RNAs specifying the negative polarity of the heterologous gene and the viral polymerase binding site using an RNA ligase.

Bicistronic mRNA could be constructed to permit internal initiation of translation of viral sequences and allow for the expression of foreign protein coding sequences from the regular terminal initiation site. Alternatively, a bicistronic mRNA sequence may be constructed wherein the viral sequence is translated from the regular terminal open reading frame, while the foreign sequence is initiated from an internal site. Certain internal ribosome entry site (IRES) sequences may be utilized. The IRES sequences which are chosen should be short enough to not interfere with Newcastle disease virus packaging limitations. Thus, it is preferable that the IRES chosen for such a bicistronic approach be no more than 500 nucleotides in length, with less than 250 nucleotides being preferred. Further, it is preferable that the IRES utilized not share sequence or structural homology with picornaviral elements. Preferred IRES elements include, but are not limited to the mammalian BiP FRES and the hepatitis C virus IRES.

Alternatively, a foreign protein may be expressed from an internal transcriptional unit in which the transcriptional unit has an initiation site and polyadenylation site. In another embodiment, the foreign gene is inserted into a negative-strand RNA virus gene such that the resulting expressed protein is a fusion protein.

5.2. EXPRESSION VECTORS ENCODING VIRAL PROTEINS

Expression vectors used to express viral proteins, in particular viral proteins for RNP complex formation, will preferably express viral proteins homologous to the desired virus. The expression of viral proteins by these expression vectors may be regulated by any regulatory sequence known to those of skill in the art. The regulatory sequence may be a constitutive promoter, an inducible promoter or a tissue-specific promoter. In a specific embodiment, the regulatory sequence comprises the adenovirus 2 major late promoter linked to the spliced tripartite leader sequence of human adenovirus 2, as described by Berg et al., Bio Techniques 14:972-978.

Promoters which may be used to control the expression of viral proteins in protein expression vectors include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the .beta.-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Omitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; KoIlias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Appropriate protein expression vectors known to those of skill in the art can be used to express the viral proteins. For example, the plasmid pGT-h described in Berg et al., BioTechniques 14:972-978 or pcDNA3 vectors can be used to construct expression vectors for viral proteins.

In a specific embodiment, the protein expression vector comprises a promoter operably linked to a nucleic acid sequence, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene). In another embodiment, a protein expression vector that is capable of producing bicistronic mRNA may be produced by inserting bicistronic mRNA sequence. Certain internal ribosome entry site (IRES) sequences may be utilized. Preferred IRES elements include, but are not limited to the mammalian BiP IRES and the hepatitis C virus IRES.

Expression vectors containing gene inserts can be identified by three general approaches: (a) nucleic acid hybridization; (b) presence or absence of "marker" gene functions; and (c) expression of inserted sequences. In the first approach, the presence of the viral gene inserted in an expression vector(s) can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to the inserted gene(s). In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., resistance to antibiotics or transformation phenotype) caused by the insertion of the gene(s) in the vector(s). In the third approach, expression vectors can be identified by assaying the gene product expressed. Such assays can be based, for example, on the physical or functional properties of the viral protein in in vitro assay systems, e.g., binding of viral proteins to antibodies.

In a specific embodiment, one or more protein expression vectors encode and express the viral proteins necessary for the formation of RNP complexes. In another embodiment, one or more protein expression vectors encode and express the viral proteins necessary to form viral particles. In yet another embodiment, one or more protein expression vectors encode and express the all of the viral proteins of a particular negative-strand RNA virus.

5.3. GENERATION OF RECOMBINANT NEGATIVE STRAND RNA VIRUSES

The present invention provides methods of generating infectious recombinant negative-strand RNA virus by introducing protein expression vectors and vRNA or corresponding cRNA expressing expression vectors into host cells in the absence of helper virus. The present invention also provides methods of generating infectious recombinant negative-strand RNA virus by introducing protein expression vectors and vRNA or corresponding cRNA expressing expression vectors into host cells in the presence of helper virus.

Protein expression vectors and expression vectors directing the expression of vRNAs or corresponding cRNAs can be introduced into host cells using techniques known to those of skill in the art. For example, expression vectors of the invention can be introduced into host cells by employing electroporation, DEAE-dextran, calcium phosphate precipitation, liposomes, microinjection, and microparticle-bombardment (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ed., 1989, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). The expression vectors of the invention may be introduced into host cells simultaneously or sequentially.

In one embodiment, one or more expression vectors directing the expression of vRNA(s) or corresponding cRNA(s) are introduced into host cells prior to the introduction of expression vectors directing the expression of viral proteins. In another embodiment, one or more expression vectors directing the expression of viral proteins are introduced into host cells prior to the introduction of the one or more expression vectors directing the expression of vRNA(s) or corresponding cRNA(s). In accordance with these embodiments, the expression vectors directing the expression of the vRNA(s) or corresponding cRNA(s) may introduced together or separately in different transfections. Further, in accordance with these embodiments, the expression vectors directing the expression of the viral proteins can be introduced together or separately in different transfections.

In another embodiment, one or more expression vectors directing the expression of vRNA(s) or corresponding cRNA(s) and one or more expression vectors directing the expression of viral proteins are introduced into host cells simultaneously. Preferably, all of the expression vectors are introduced into host cells using liposomes.

Appropriate amounts and ratios of the expression vectors for carrying out a method of the invention may be determined by routine experimentation. As guidance, in the case of liposomal transfection or calcium precipitation of plasmids into the host cells, it is envisaged that each plasmid may be employed at a few .mu.gs, e.g., 1 to 10 .mu.g, for example, diluted to a final total DNA concentration of about 0.1 .mu.g/ml prior to mixing with transfection reagent in conventional manner. It may be preferred to use vectors expressing NP and/or RNA-dependent RNA polynerase subunits at a higher concentration than those expressing vRNA segments. One skilled in the art will appreciate that the amounts and ratios of the expression vectors may vary depending upon the host cells.

In one embodiment, at least 0.5 .mu.g, preferably at least 1 .mu.g, at least 2.5 .mu.g, at least 5 .mu.g, at least 8 .mu.g, at least 10 .mu.g, at least 15 .mu.g, at least 20 .mu.g, at least 25 .mu.g, or at least 50 .mu.g of one or more protein expression vectors of the invention are introduced into host cells to generate infectious recombinant negative-strand RNA virus. In another embodiment, at least 0.5 .mu.g, preferably at least 1 .mu.g, at least 2.5 .mu.g, at least 5 .mu.g, at least 8 .mu.g, at least 10 .mu.g, at least 15 .mu.g, at least 20 .mu.g, at least 25 .mu.g or at least 50 .mu.g of one or more expression vectors of the invention directing the expression of vRNAs or cRNAs are introduced into host cells to generate infectious recombinant negative-strand RNA virus.

Host cells which may be used to generate the negative-strand RNA viruses of the invention include primary cells, cultured or secondary cells, and transformed or immortalized cells (e.g., 293 cells, 293T cells, CHO cells, Vero cells, PK, MDBK, OMK and MDCK cells). Host cells are preferably animal cells, more preferably mammalian cells, and most preferably human cells. In a preferred embodiment, infectious recombinant negative-strand RNA viruses of the invention are generated in 293T cells.

It is known that Vero cells are deficient in interferon expression (Diaz et al., 1998, Proc. Natl. Acad. Sci. USA 85:5259-5263), which might be a factor in attaining good viral rescue. Hence, it is extrapolated that Vero cells and other cells deficient in interferon activity or response which will support growth of segmented negative-strand RNA viruses may be useful in the practice of the invention.

In order to rescue recombinant influenza B viruses, 293 T cells may not be the most efficient host cell to achieve rescue. Thus, in accordance with the present invention, methods to achieve rescue influenza B virus should utilize host cells which support the efficient replication of influenza B, such as MDCK (canine kidney), PK (porcine kidney) or OMK (owl monkey kidney) cells. Alternatively, MDBK (bovine kidney) cells may be used as hots cells to support rescue of influenza B. Despite the fact that MDBK cells do not support the growth of influenza B, using a reverse genetics approach this cell line supports rescue of influenza B (Barclay et al., 1995, J. Virol. 69:1275-1279).

The present invention provides methods of generating infectious recombinant negative-strand RNA virus in stably transduced host cell lines. The stably transduced host cell lines of the invention may be produced by introducing cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker into host cells. Following the introduction of the foreign DNA, the transduced cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker confers resistance to the cells and allows the cells to stably integrate the DNA into their chromosomes. Transduced host cells with the DNA stably integrated can be cloned and expanded into cell lines.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147) genes.

The infectious recombinant negative-strand RNA viruses generated by methods of the invention which are not attenuated, may attenuated or killed by, for example, classic methods. For example, recombinant negative-strand RNA viruses of the invention may be killed by heat or formalin treatment, so that the virus is not capable of replicating. Recombinant negative-strand RNA viruses of the invention which are not attenuated may be attenuated by, e.g., passage through unnatural hosts to produce progeny viruses which are immunogenic, but not pathogenic.

Attenuated or killed viruses produced in accordance with the invention may subsequently be incorporated into a vaccine composition in conventional manner. Where such a virus has a chimeric vRNA segment as discussed above which encodes a foreign antigen, it may be formulated to achieve vaccination against more than one pathogen simultaneously. Attenuated recombinant viruses produced in accordance with the invention which possess a chimeric vRNA segment may also be designed for other therapeutic uses, e.g., an anti-tumor agent or gene therapy tool, in which case production of the virus will be followed by its incorporation into an appropriate pharmaceutical composition together with a pharmaceutically acceptable carrier or diluent.

Helper virus free rescue in accordance with the invention is particularly favored for generation of reassortant viruses, especially reassortant influenza viruses desired for vaccine use. For example, by means of viral rescue in accordance with the invention the HA and NA vRNA segments of an influenza virus, e.g., influenza A/PR8/34 which is recognized as suitable for human administration, may be readily substituted with the HA and NA vRNA segments of an influenza strain associated with aninfluenza infection epidemic. Such reassortant influenza viruses may, for example, be used for production of a killed influenza vaccine in conventional manner.

The methods of the present invention may be modified to incorporate aspects of methods known to those skilled in the art, in order to improve efficiency of rescue of infectious viral particles. For example, the reverse genetics technique involves the preparation of synthetic recombinant viral RNAs that contain the non-coding regions of the negative strand virus RNA which are essential for the recognition by viral polymerases and for packaging signals necessary to generate a mature virion. The recombinant RNAs are synthesized from a recombinant DNA template and reconstituted in vitro with purified viral polymerase complex to form recombinant ribonucleoprotein (RNPs) which can be used to transect cells. A more efficient transfection is achieved if the viral polymerase proteins are present during transcription of the synthetic RNAs either in vitro or in vivo. The synthetic recombinant RNPs can be rescued into infectious virus particles. The foregoing techniques are described in U.S. Pat. No. 5,166,057 issued Nov. 24, 1992; in U.S. Pat. No. 5,854,037 issued Dec. 29, 1998; in U.S. Pat. No. 5,789,229 issued Aug. 4, 1998; in European Patent Publication EP 0702085A1, published Feb. 20, 1996; in U.S. Pat. application Ser. No. 09/152,845; in International Patent Publications PCR WO97/12032 published Apr. 3, 1997; WO96/34625 published Nov. 7, 1996; in European Patent Publication EP-A780475; WO99/02657 published Jan. 21, 1999; WO98/53078 published Nov. 26, 1998; WO98/02530 published Jan. 22, 1998; WO99/15672 published Apr. 1, 1999; WO98/13501 published Apr. 2, 1998; WO97/06720 published Feb. 20, 1997; and EPO 780 47SA1 published Jun. 25, 1997, each of which is incorporated by reference herein in its entirety.

5.4. SEGMENTED NEGATIVE-STRAND RNA VIRUS EMBODIMENTS

The present invention provides a method for generating in cultured cells infectious viral particles of a segmented negative-strand RNA virus having greater than 3 genomic vRNA segments, for example an influenza virus such as an influenza A virus, said method comprising: (a) providing a first population of cells capable of supporting growth of said virus and having introduced a first set of expression vectors capable of directly expressing in said cells genomic vRNA segments to provide the complete genomic vRNA segments of said virus, or the corresponding cRNAs, in the absence of a helper virus to provide any such RNA segment, said cells also being capable of providing a nucleoprotein and RNA-dependent RNA polymerase whereby RNP complexes containing the genomic vRNA segments of said virus can be formed and said viral particles can be assembled within said cells; and (b) culturing said cells whereby said viral particles are produced.

The present invention also provides a method for generating in cultured cells infectious viral particles of a segmented negative-strand RNA virus, said method comprising: (i) providing a first population of cells which are capable of supporting the growth of said virus and which are modified so as to be capable of providing (a) the genomic vRNAs of said virus in the absence of a helper virus and (b) a nucleoprotein and RNA-dependent RNA polymerase whereby RNA complexes containing said genomic vRNAs can be formed and said viral particles can be assembled, said genomic vRNAs being directly expressed in said cells under the control of a human Pol I promoter or functional derivative thereof: and (ii) culturing said cells whereby said viral particles are produced.

The present specification also provides a method for generating in cultured cells infectious viral particles of a segmented negative-strand RNA virus, said method comprising: (i) providing a population of cells which are capable of supporting the growth of said virus and which are modified so as be capable of providing (a) the genomic vRNAs of said virus in the absence of a helper virus and (b) a nucleoprotein and RNA-dependent RNA polymerase whereby RNP complex or complexes containing said genomic vRNAs can be formed and said viral particles can be assembled, said genomic RNAs being directly expressed in said cells under the control of a mammalian Pol I, Pol II or Pol III promoter or a functional derivative thereof, e.g., the truncated human Pol I promoter as previously noted above; and (ii) culturing said cells whereby said viral particles are produced.

In a specific embodiment, an infectious recombinant negative-strand RNA virus having, at least 4, preferably at least 5, at least 6, or at least 7 genomic vRNA segments in a host cell using the methods described herein.

In a preferred embodiment, the present invention provides for methods of generating infectious recombinant influenza virus in host cells using expression vectors to express the vRNA segments or corresponding cRNAs and influenza virus proteins, in particular PB1, PB2, PA and NA. In accordance with this embodiment, helper virus may or may not be included to generate the infectious recombinant influenza viruses.

The infectious recombinant influenza viruses of the invention may or may not replicate and produce progeny. Preferably, the infectious recombinant influenza viruses of the invention are attenuated. Attenuated infectious recombinant influenza viruses may, for example, have a mutation in the NS1 gene.

In a preferred embodiment, the infectious recombinant influenza viruses of the invention express heterologous (i.e., non-influenza virus) sequences. In another embodiment, the infectious recombinant influenza viruses of the invention express influenza virus proteins from different influenza strains. In yet another preferred embodiment, the infectious recombinant influenza viruses of the invention express fusion proteins.

5.5. NEWCASTLE DISEASE VIRUS EMBODIMENTS

A specific embodiment of the present invention is the Applicants' identification of the correct nucleotide sequence of the 5' and 3' termini of the negative-sense genomes RNA of NDV. The nucleotide sequence of the 3' termini of the NDV negative-sense genome RNA of the present invention differs significantly from the NDV 3' termini sequence previously disclosed by Collins et al. in Fundamental Virology 3rd Ed. 1996 by Lippincott-Raven Publishers as shown in FIG. 6. The identification of the correct nucleotide sequence of the NDV 3' termini allows for the first time the engineering of recombinant NDV RNA templates, the expression of the recombinant RNA templates and the rescue of recombinant NDV particles.

Heterologous gene coding sequences flanked by the complement of the viral polymerase binding site/promoter, e.g, the complement of 3'-NDV virus terminus of the present invention, or the complements of both the 3'- and 5'-NDV virus termini may be constructed using techniques known in the art. The resulting RNA templates may be of the negative-polarity and contain appropriate terminal sequences which enable the viral RNA-synthesizing apparatus to recognize the template. Alternatively, positive-polarity RNA templates which contain appropriate terminal sequences which enable the viral RNA-synthesizing apparatus to recognize the template, may also be used. Recombinant DNA molecules containing these hybrid sequences can be cloned and transcribed by a DNA-dependent RNA polymerase, such as bacteriophage T7, T3, or the SP6 polymerase and the like, to produce in vitro and in vivo the recombinant RNA templates which possess the appropriate viral sequences that allow for viral polymerase recognition and activity.

As described above, heterologous sequences can be: 1) antigens that are characteristic of a pathogen; 2) antigens that are characteristic of autoimmune disease; 3) antigens that are characteristic of an allergen; and 4) antigens that are characteristic of a tumor. The heterologous sequences can be introduced into viral nucleic acid sequences by techniques described herein or known to those of skill in the art.

The gene segments coding for the NDV HN, P, NP, M, F, or L proteins may be used for the insertion of heterologeous gene products. Insertion of a foreign gene sequence into any of these segments could be accomplished by either a complete replacement of the viral coding region with the foreign gene or by a partial replacement. Complete replacement would probably best be accomplished through the use of PCR-directed mutagenesis. Briefly, PCR-primer A would contain, from the 5' to 3' end: a unique restriction enzyme site, such as a class IIS restriction enzyme site (i.e., a "shifter" enzyme; that recognizes a specific sequence but cleaves the DNA either upstream or downstream of that sequence); a stretch of nucleotides complementary to a region of the NDV gene; and a stretch of nucleotides complementary to the carboxy-terminus coding portion of the foreign gene product. PCR-primer B would contain from the 5' to 3' end: a unique restriction enzyme site; a stretch of nucleotides complementary to a NDV gene; and a stretch of nucleotides corresponding to the 5' coding portion of the foreign gene. After a PCR reaction using these primers with a cloned copy of the foreign gene, the product may be excised and cloned using the unique restriction sites. Digestion with the class IIS enzyme and transcription with the purified phage polymerase would generate an RNA molecule containing the exact untranslated ends of the NDV gene with a foreign gene insertion. In an alternate embodiment, PCR-primed reactions could be used to prepare double-stranded DNA containing the bacteriophage promoter sequence, and the hybrid gene sequence so that RNA templates can be transcribed directly without cloning.

The hemagglutinin and neuraminidase activities of NDV are coded for by a single gene, HN. The HN protein is a major surface glycoprotein of the virus. For a variety of viruses, such as influenza, the hemagglutinin and neuraminidase proteins have been demonstrated to contain a number of antigenic sites. Consequently, this protein is a potential target for the humoral immune response after infection. Therefore, substitution of antigenic sites within HN with a portion of a foreign protein may provide for a vigorous humoral response against this foreign peptide. If a sequence is inserted within the HN molecule and it is expressed on the outside surface of the HN it will be immunogenic. For example, a peptide derived from gp160 of HrV could be inserted into antigenic site of the HN protein for antigenic presentation by the chimeric virus, resulting in the elicitation of both a humoral immune response. In a different approach, the foreign peptide sequence may be inserted within the antigenic site without deleting any viral sequences. Expression products of such constructs may be useful in vaccines against the foreign antigen, and may indeed circumvent a problem discussed earlier, that of propagation of the recombinant virus in the vaccinated host. An intact HN molecule with a substitution only in antigenic sites may allow for HN function and thus allow for the construction of a viable virus. Therefore, this virus can be grown without the need for additional helper functions. The virus may also be attenuated in other ways to avoid any danger of accidental escape.

Other hybrid constructions may be made to express proteins on the cell surface or enable them to be released from the cell. As a surface glycoprotein, the HN has an amino-terminal cleavable signal sequence necessary for transport to the cell surface, and a carboxy-terminal sequence necessary for membrane anchoring. In order to express an intact foreign protein on the cell surface it may be necessary to use these HN signals to create a hybrid protein. In this case, the fusion protein may be expressed as a separate fusion protein from an additional internal promoter. Alternatively, if only the transport signals are present and the membrane anchoring domain is absent, the protein may be secreted out of the cell.

The recombinant templates prepared as described above can be used in a variety of ways to express the heterologous gene products in appropriate host cells or to create chimeric viruses that express the heterologous gene products. In one embodiment, the recombinant template can be used to transect appropriate host cells, may direct the expression of the heterologous gene product at high levels. Host cell systems which provide for high levels of expression include continuous cell lines that supply viral functions such as cell lines superinfected with NDV, cell lines engineered to complement NDV finction, etc.

In an alternate embodiment of the invention, the recombinant templates may be used to transect cell lines that express a viral polymerase protein in order to achieve expression of the heterologous gene product. To this end, transformed cell lines that express a polymerase protein such as the L protein may be utilized as appropriate host cells. Host cells may be similarly engineered to provide other viral functions or additional functions such as NP or HNT.

In another embodiment, a helper virus may provide the RNA polymerase protein utilized by the cells in order to achieve expression of the heterologous gene product.

In yet another embodiment, cells may be transfected with vectors encoding viral proteins such as the NP, P and L proteins. Examples of such vectors are illustrated in FIG. 3A-3C.

In order to prepare chimeric virus, containing modified NDV virus RNAs or RNA coding for foreign proteins in the plus or minus sense, may be used to transect cells which are also infected with a "parent" NDV virus. Following reassortment, the novel viruses may be isolated and their genomes be identified through hybridization analysis. In additional approaches described herein the production of infectious chimeric virus may be replicated in host cell systems that express an NDV viral polymerase protein (e.g., in virusihost cell expression systems; transformed cell lines engineered to express a polymerase protein, etc.), so that infectious chimeric virus are rescued. In this instance, helper virus need not be utilized since this function is provided by the viral polymerase proteins expressed.

In a particularly desirable approach, cells engineered to express all NDV viral genes may result in the production of infectious chimeric virus which contain the desired genotype; thus eliminating the need for a selection system. Theoretically, one can replace any one of the six genes or part of any one of the six genes of NDV with a foreign sequence. However, a necessary part of this equation is the ability to propagate the defective virus (defective because a normal viral gene product is missing or altered). A number of possible approaches exist to circumvent this problem. In one approach a virus having a mutant protein can be grown in cell lines which are constricted to constitutively express the wild type version of the same protein. By this way, the cell line complements the mutation in the virus. Similar techniques may be used to construct transformed cell lines that constitutively express any of the NDV genes. These cell lines which are made to express the viral protein may be used to complement the defect in the recombinant virus and thereby propagate it. Alternatively, certain natural host range systems may be available to propagate recombinant virus.

A third approach to propagating the recombinant virus may involve co-cultivation with wild-type virus. This could be done by simply taking recombinant virus and co-infecting cells with this and another wild-type virus (preferably a vaccine strain). The wild-type virus should complement for the defective virus gene product and allow growth of both the wild-type and recombinant virus. Alternatively, a helper virus may be used to support propagation of the recombinant virus.

In another approach, synthetic templates may be replicated in cells co-infected with recombinant viruses that express the NDV virus polymerase protein. In fact, this method may be used to rescue recombinant infectious virus in accordance with the invention. To this end, the an NDV polymerase protein may be expressed in any expression vector/host cell system, including but not limited to viral expression vectors (e.g., vaccinia virus, adenovirus, baculovirus, etc.) or cell lines that express a polymerase protein (e.g., see Krystal et al., 1986, Proc. Natl. Acad. Sci. USA 83: 2709-2713). Moreover, infection of host cells expressing all six NIDV proteins may result in the production of infectious chimeric virus particles. This system would eliminate the need for a selection system, as all recombinant virus produced would be of the desired genotype.

It should be noted that it may be possible to construct a recombinant virus without altering virus viability. These altered viruses would then be growth competent and would not need helper functions to replicate. For example, alterations in the hemagglutinin neuraminidase gene discussed, sitpia, may be used to construct such viable chimeric viruses.

5.6. PURIFICATION/ISOLATION OF RECOMBINANT NEGATIVE STRAND RNA VIRUSES

The recombinant negative strand RNA viruses of the invention can be isolated or purified using techniques known to those of skill in the art (see, e.g., U.S. Pat. No. 5,948,410 and R. J. Kuchler, "Biochemical Methods in Cell Culture and Virology", Dowden, Hutchinson and Ross, Inc., Stroudsburg, Pa. (1977)). For example, using one isolation method, supernatant from host cells expressing the recombinant negative-strand RNA viruses of the invention are filtered through a depth filter with a nominal pore size of 0.5 micron to remove the cellular debris. Subsequently, the recombinant negative-strand RNA viruses are concentrated and purified by ultrafiltration using a membrane with a molecular weight cut-off. Sucrose is added to the concentrate to a final concentration of 30% (w/v) after which formaldehyde is added to a final concentration of 0.015% (w/v). This mixture is stirred at 2-8^oC. for 72 hours. Next the virus concentrate is diluted five-fold with phosphate buffered saline and loaded onto a affinity column containing Amicon Cellufine Sulphate. After removing impurities by washing with phosphate buffered saline the virus is eluted with a solution of 1.5 molar sodium chloride in phosphate buffered saline. The eluate is concentrated and desalted by ultrafiltration using a membrane with a molecular weight cut-off.

In another isolation method, supernatant from host cells expressing the recombinant negative-strand RNA viruses of the invention is subject to centrifugation at a speed which will not pellet the virus (e.g., 2,500 rpm for about 20 minutes). The supernatant may then be further purified by ultrafiltration employing a filter having a pore size that is larger than the viral particles. Preferably, a filter of approximately 0.22 microns is used. Following filtration, the viral particles are collected by polyethylene glycol precipitation followed by centriflgation or, more preferably, by high speed centrifugation at about 70,000 rpm. The viral particles are then resuspended in a small volume of buffer, preferably TNE (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.4). A non-ionic detergent may optionally be added to the viral particle suspension to dissolve any contaminants. Although the high speed viral pellet is sufficiently pure to use as a source of viral RNA the viral suspension may optionally be further purified by sucrose density gradient centrifugation.

An "isolated" or "purified" recombinant negative-strand RNA virus is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, and is substantially free of contaminating viruses (e.g., helper virus). A recombinant negative-strand RNA virus that is substantially free of cellular material includes preparations of the recombinant negative-strand RNA virus is at least 50%, preferably at least 60%, at least 75%, at least 85%, at least 95%, or at least 99% free of heterologous protein (also referred to herein as a "contaminating protein"). A recombinant negative-strand RNA virus that is substantially free of contaminating virus includes preparations of the recombinant negative-strand RNA virus is at least 50%, preferably at least 60%, at least 75%, at least 85%, at least 95%, or at least 99% free of contaminating viruses.

5.7. ASSAYS FOR THE IDENTIFICATION OF RECOMBINANT NEGATIVE STRAND RNA VIRUSES

The production of the recombinant negative-strand RNA viruses of the invention may assessed using any technique known to one of skill in the art. For example, recombinant negative-strand RNA viruses of the invention may be assessed by cell-free reverse transcriptase (hereinafter "RT") activity assay in the cultures and by electron microscopy. Further, any conventional assay which detects virus-specific proteins may be employed to detect the production of the recombinant negative-strand RNA viruses of the invention. Such assays include, for example, Western blots, ELISA, radioimmunoassay, or polyacrylamide gel electrophoresis and comparison to a virus standard.

The production of infectious, replicating recombinant negative-strand RNA viruses of the invention may be assessed using techniques known to those of skill in the art. In particular, the production of infectious, replicating recombinant negative-strand RNA viruses of the invention may be assessed by a plaque assay using, for example, MDCK cells.

5.8. VACCINE FORMULATIONS

Virtually any heterologous gene sequence may be constructed into the viruses of the invention for use in vaccines. Preferably, epitopes that induce a protective immune response to any of a variety of pathogens, or antigens that bind neutralizing antibodies may be expressed by or as part of the viruses. For example, heterologous gene sequences that can be constructed into the viruses of the invention for use in vaccines include but are not limited to epitopes of human immunodeficiency virus (HIV) such as gp120; hepatitis B virus surface antigen (HBsAg); the glycoproteins of herpes virus (e.g. gD, gE); VP1 of poliovirus; antigenic determinants of non-viral pathogens such as bacteria and parasites, to name but a few. In another embodiment, all or portions of immunioglobulin genes may be expressed. For example, variable regions of anti-idiotypic immunoglobulins that mimic such epitopes may be constructed into the viruses of the invention.

Either a live recombinant viral vaccine or an inactivated recombinant viral vaccine can be formulated. A live vaccine may be preferred because multiplication in the host leads to a prolonged stimulus of similar kind and magnitude to that occurring in natural infections, and therefore, confers substantial, long-lasting immunity. Production of such live recombinant virus vaccine formulations may be accomplished using conventional methods involving propagation of the virus in cell culture or in the allantois of the chick embryo followed by purification.

Vaccine formulations may include genetically engineered negative strand RNA viruses that have mutations in the NS1 or analogous gene. They may also be formulated using negative strand RNA viruses that have mutations in the NS1 or analogous gene that are natural variants, such as the A/turkey/Ore/71 natural variant of influenza A, or B/201, and AWBY-234, which are natural variants of influenza B. Furthermore, vaccines can include viruses that have mutations in the NS1 or analogous gene resulting from spontaneous mutation events, UV irradiation, exposure to chemical mutagens, or any other genetically-altering event.

Many methods may be used to introduce the vaccine formulations described above, these include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes. It may be preferable to introduce the virus vaccine formulation via the natural route of infection of the pathogen for which the vaccine is designed. Where a live virus vaccine preparation is used, it may be preferable to introduce the formulation via the natural route of infection for influenza virus. The ability of influenza virus to induce a vigorous secretory and cellular immune response can be used advantageously. For example, infection of the respiratory tract by influenza viruses may induce a strong secretory immune response, for example in the urogenital system, with concomitant protection against a particular disease causing agent.

5.9. PHARMACEUTICAL COMPOSITIONS

The present invention encompasses pharmaceutical compositions comprising recombinant viruses of the invention to be used as anti-viral agents or anti-tumor agents. The pharmaceutical compositions have utility as an anti-viral prophylactic and thus in accordance may be administered to a subject when the subject has been exposed or is expected to be exposed to a virus. For example, in the event that a child comes home from school where he is exposed to several classmates with the flu, a parent would administer the anti-viral pharmaceutical composition of the invention to herself, the child and other family members to prevent viral infection and subsequent illness.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, in a preferred embodiment in a preferred embodiment it may be desirable to introduce the pharmaceutical compositions of the invention into the lungs by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.

In another embodiment, the pharmaceutical composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (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, N.Y. (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, i.e., the lung, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a mutant virus, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified fonn, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the pharmaceutical composition of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from ill vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral fonmulations preferably contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In a preferred embodiment, the kit contains a Therapeutic of the invention, e.g., a lats protein, or therapeutically effective lats derivative or analog, or nucleic acid encoding the same, and one or more chemotherapeutic agents.

Claim 1 of 49 Claims

What is claimed is:

1. A method for rescuing a chimeric recombinant negative strand RNA virus, wherein said chimeric virus expresses heterologous nucleic acid sequences, comprising:

(a) introducing into a 293T cell, expression vectors which direct the expression in said cells of genomic or antigenomic vRNA segments, and a nucleoprotein, and an RNA-dependent RNA polymerase, so that ribonucleoprotein complexes can be formed and viral particles can be assembled in the absence of helper virus; and

(b) culturing said cells wherein viral particles are packaged and rescued.

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