Infectious recombinant paramyxovirus from antigenomic cDNA
AssigneeCrucell Switzerland AG
InventorsMartin A. Billeter, Pius Spielhofer, Karin Kälin, Frank Radecke, Henriette Schneider
Life of this patent
Estimating the term…
Abstract
The present invention relates, in general, to a methodology or the generation of 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.
Claims14
An isolated batch of infectious RNA viruses for use as a vaccine, said infectious RNA viruses each comprising:
- (a) the entire (−)-strand sequence of a non-segmented negative-strand RNA virus of the family Paramyxoviridae which obeys the rule of six;
- operatively linked to
- (b) a foreign expressible RNA fragment,
- wherein the length of the genome of the infectious RNA virus is an integral multiple of six,
- and further wherein the infectious RNA virus is obtainable by the steps of:
- (1) introducing into a host cell a plasmid comprising a cDNA molecule, said cDNA molecule comprising the entire (+)-strand sequence of said negative-strand RNA virus operatively linked to an expression control sequence, which allows the synthesis of antigenomic RNA transcripts bearing the authentic 3′-termini without help of a helper virus, said host cell comprising (i) transfected nucleic acid molecules containing nucleic acid sequences that allow expression of an exogenous RNA polymerase, a viral N protein and a viral P protein, and (ii) a nucleic acid molecule containing nucleic acid sequence encoding a viral L protein;
- and (2) recovering and isolating the infectious RNA virus, wherein the isolated batch of infectious RNA viruses is free of helper virus.
An isolated batch of infectious RNA viruses for use as a vaccine, said infectious RNA viruses each comprising:
- (a) the entire (−)-strand sequence of a non-segmented negative-strand RNA virus of the family Paramyxoviridae which obeys the rule of six;
- operatively linked to
- (b) a foreign expressible RNA fragment,
- wherein the length of the genome of the infectious RNA virus is an integral multiple of six,
- and further wherein the infectious RNA virus is obtainable by the steps of:
- (1) introducing into a host cell a plasmid comprising a cDNA molecule, said cDNA molecule comprising the entire (+)-strand sequence of said negative-strand RNA virus operatively linked to an expression control sequence, which allows the synthesis of antigenomic RNA transcripts bearing the authentic 3′-termini without help of a helper virus, said host cell comprising (i) stably transfected nucleic acid molecules containing nucleic acid sequences that allow genomic expression of an exogenous RNA polymerase, a viral N protein and a viral P protein, and (ii) a nucleic acid molecule containing nucleic acid sequence encoding a viral L protein;
- and (2) recovering and isolating the infectious RNA virus,
- wherein the isolated batch of infectious RNA viruses is free of helper virus.
Dependencies are inferred from the claim text.