Host cells comprising cDNA corresponding to the antigenome of nonsegmented negative strand RNA viruses, further encoding antigenically active proteins
The present invention relates, in general, to a methodology for the generation or nonsegmented negative-strand RNA viruses (Pringle, 1991) from cloned deoxyribonucleic acid (cDNA). Such rescued viruses are suitable for use as vaccines, or alternatively, as plasmids in somatic gene therapy applications. The invention also relates to cDNA molecules suitable as tools in this methodology and to helper cell lines allowing the direct rescue of such viruses. Measles virus (MV) is used as a model for other representatives of the Mononegavirales, in particular the family Paramyxoviridae.
1. An isolated eukaryotic host cell comprising (1) stably transfected expression plasmids that allow genomic expression of an exogenous RNA polymerase, a viral N protein, and a viral P protein in the host cell; (2) a nucleic acid sequence encoding a viral L protein; and (3) a cDNA molecule, said cDNA molecule comprising:
(a) a (+) strand sequence of a non-segmented negative-strand RNA virus of the family Paramyxoviridae from which RNA transcripts bearing the authentic 3′ termini can be transcribed; and
(b) an expression control sequence,
wherein said (+) strand sequence of a non-segmented negative-strand RNA virus is operatively linked to the expression control sequence, and wherein the host cell, without a replicating vaccinia helper virus, produces an infectious RNA virus containing the (−) strand sequence of the non-segmented negative-strand RNA virus of the family Paramyxoviridae, said (−) strand sequence of the non-segmented negative-strand RNA virus obeying the rule of six.
2. The host cell of claim 1 , wherein the nucleic acid encoding the viral L protein is stably transfected into the host cell, said stable transfection allowing genomic expression of the viral L protein in the host cell.
3. The host cell of claim 1 , wherein the nucleic acid sequence encoding the viral L protein is derived from measles or mumps virus, and wherein the viral N and P proteins are encoded by nucleic acid sequences derived from measles or mumps virus.
4. The host cell of claim 3 , wherein the non-segmented negative-strand RNA virus is measles virus.
5. The host cell of claim 3 , wherein the non-segmented negative-strand RNA virus is mumps virus.
6. The host cell of claim 1 , wherein the expression control sequence comprises an RNA polymerase promoter.
7. The host cell of claim 6 , wherein the RNA polymerase promoter is a heterologous RNA polymerase promoter, with respect to the non-segmented negative-strand RNA virus.
8. The host cell of claim 1 , wherein the (+) strand sequence of the non-segmented negative-strand RNA virus contains a sequence variation, with respect to a wild-type non-segmented negative-strand RNA virus.
9. The host cell of claim 8 , wherein the sequence variation is a deletion.
10. The host cell of claim 1 , further comprising an expressible DNA fragment inserted into said (+) strand sequence of a non-segmented negative-strand RNA virus.
11. The host cell of claim 10 , wherein the expressible DNA fragment is inserted into a non-coding region of said (+) strand sequence of a non-segmented negative-strand RNA virus.
12. The host cell of claim 11 , wherein the expressible DNA fragment is inserted in the 5′ terminal region of the (+) strand sequence of a non-segmented negative-strand RNA virus.
13. The host cell of claim 12 , wherein said expressible DNA fragment is homologous or heterologous with respect to the non-segmented negative-strand RNA virus.
14. The host cell of claim 13 , wherein the expressible DNA fragment encodes at least one immunogenic epitope.
15. The host cell of claim 14 , wherein said expressible DNA fragment encodes an immunogenic epitope of an envelope protein of a pathogen.
16. The host cell of claim 10 , wherein said expressible DNA fragment is derived from a virus, a bacterium, a pathogen, or a parasite.
17. The host cell of claim 10 , wherein said expressible DNA fragment encodes a toxin.
18. The host cell of claim 10 wherein the expressible DNA fragment is inserted into a region of said (+) strand sequence encoding a viral protein, said insertion being effected in a manner maintaining the reading frame to create a fusion protein, and permitting the expression of said fragment under the control of the promoter of the viral protein.
19. The host cell of claim 18 wherein the expressible fragment encodes an immunogenic epitope.
20. The host cell of claim 10 , wherein the expressible DNA fragment is inserted upstream of a viral protein coding region to avoid formation of a fusion protein, but allowing expression of the downstream viral protein coding region either by a stop/restart mechanism where the last A residue of the upstream termination triplet coincides with that of the start codon of the downstream coding region, or by placing an internal ribosome entry site (IRES) between the two coding regions.