DNA-transfection system for the generation of infectious influenza virus

A composition comprising a negative strand RNA virus virion, wherein viral internal proteins of the virion are from a virus strain well adapted to grow in culture or from an attenuated strain, or both and viral antigen proteins, of the virion are from a pathogenic virus strain, wherein said viral internal proteins and said viral antigen proteins are generated by a minimum plasmid-based system comprising a set of plasmids wherein each plasmid comprises one viral genomic segment, and wherein the viral cDNA corresponding to the viral genomic segment is inserted between an RNA polymerase I (pol I) promoter and a regulatory element, which results in expression of vRNA or cRNA with an exact 3′ end, which are in turn inserted between an RNA polymerase II (pol II) promoter and a polyadenylation signal, which results in expression of viral mRNA and a corresponding viral protein, wherein the expression of vRNAs or cRNAs and viral internal proteins and antigen proteins from said set of plasmids results in a negative strand RNA virus virion, which is well adapted to grow in culture or is attenuated or both and is pathogenic.

A method of producing a composition comprising negative strand RNA virus virion, which comprises viral internal proteins from a virus that is attenuated or grows well in culture and viral antigen proteins from a pathogenic virus, which method comprises

• (1) selecting viral genomic segments encoding said viral internal proteins from a virus that grows well in culture or is attenuated and viral genomic segments encoding said viral antigen proteins from a pathogenic virus;
• (2) introducing in a host cell a set of plasmids, wherein each plasmid comprises one viral genomic segment of said viral genomic segments, and wherein the viral cDNA corresponding to the viral genomic segment is inserted between an RNA polymerase I (pol I) promoter and a regulatory element, which results in expression of vRNA or cRNA with an exact 3′ end, which are in turn inserted between an RNA polymerase II (pol II) promoter and a polyadenylation signal, which results in expression of viral mRNA and a corresponding viral protein, and
• (3) culturing said host cell under conditions that permit production of viral internal proteins, viral antigen proteins, and vRNA or cRNA.

A method of preparing a negative strand RNA virus specific vaccine, which method comprises purifying a composition which comprises a negative strand RNA virus virion, wherein viral internal proteins of the virion are from a virus strain well adapted to grow in culture or from an attenuated strain, or both and viral antigen proteins, of the virion are from a pathogenic virus strain, wherein said negative strand RNA virus is generated culturing in a host cell a minimum plasmid-based system comprising a set of plasmids wherein each plasmid comprises one viral genomic segment, and wherein the viral cDNA corresponding to the viral genomic segment is inserted between an RNA polymerase I (pol I) promoter and a regulatory element, which results in expression of vRNA or cRNA with an exact 3′ end, which are in turn inserted between an RNA polymerase II (pol II) promoter and a polyadenylation signal, which results in expression of viral mRNA and a corresponding viral protein, wherein the expression of vRNAs or cRNAs and viral internal proteins and antigen proteins from said set of plasmids results in a negative strand RNA virus virion.

The method of 73, wherein said virion is inactivated prior to the clarification of allantoic fluid from virus.

A method of vaccinating a subject in need thereof against a negative strand RNA virus infection, which method comprises administering a negative strand RNA virus specific vaccine which comprises a negative strand RNA virus virion, wherein viral internal proteins of the virion are from a virus strain well adapted to grow in culture or from an attenuated strain, or both and viral antigen proteins, of the virion are from a pathogenic virus strain, wherein said negative strand RNA virus is generated culturing in a host cell a minimum plasmid-based system comprising a set of plasmids wherein each plasmid comprises one viral genomic segment, and wherein the viral cDNA corresponding to the viral genomic segment is inserted between an RNA polymerase I (pol I) promoter and a regulatory element, which results in expression of vRNA or cRNA with an exact 3′ end, which are in turn inserted between an RNA polymerase II (pol II) promoter and a polyadenylation signal, which results in expression of viral mRNA and a corresponding viral protein, wherein the expression of vRNAs or cRNAs and viral internal proteins and antigen proteins from said set of plasmids results in a negative strand RNA virus virion.