DNA-transfection system for the generation of infectious influenza virus
The present invention is based on the development of a dual promoter system (preferably a RNA pol I-pol II system) for the efficient intracellular synthesis of viral RNA. The resultant minimal plasmid-based system may be used to synthesize any RNA virus, preferably viruses with a negative single stranded RNA genome. The viral product of the system is produced when the plasmids of the system are introduced into a suitable host cell. One application of the system is production of attenuated, reassortant influenza viruses for use as antigens in vaccines. The reassortant viruses generated by cotransfection of plasmids may comprise genes encoding the surface glycoproteins hemagglutinin and neuraminidase from an influenza virus currently infecting the population and the internal genes from an attenuated influenza virus. An advantageous property of the present invention is its versatility; the system may be quickly and easily adapted to synthesize an attenuated version of any RNA virus. Attenuated or inactivated RNA viruses produced by the present invention may be administered to a patient in need of vaccination by any of several routes including intranasally or intramuscularly.
1 - 41 . (canceled)
42 . 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.
43 . The composition of claim 42 , wherein the regulatory element for the synthesis of vRNA or cRNA with an exact 3′ end is an RNA polymerase I (pol I) terminator sequence.
44 . The composition of claim 42 , wherein the regulatory element for the synthesis of vRNA or cRNA with an exact 3′ end is a ribozyme sequence.
45 . The composition of claim 43 , wherein the pol I promoter is proximal to the polyadenylation signal and the pol I terminator sequence is proximal to the pol II promoter.
46 . The composition of claim 43 , wherein the pol I promoter is proximal to the pol II promoter and the pol I terminator sequence is proximal to the polyadenylation signal.
47 . The composition of claim 42 , wherein said negative strand RNA virus virion is an influenza virus.
48 . The composition of claim 47 , wherein said influenza virus grows well in culture and pathogenic.
49 . The composition of claim 47 , wherein said influenza virus is attenuated and pathogenic.
50 . The composition of claim 47 , wherein said influenza virus grows well in culture, is attenuated, and is pathogenic.
51 . The composition of claim 49 , wherein said influenza virus is temperature sensitive.
52 . The composition of claim 51 , wherein said attenuated influenza virus is cold adapted.
53 . The composition of claim 48 , wherein said culture is a chicken egg or a host cell.
54 . The composition of claim 42 , wherein said viral internal proteins are selected from the group consisting of a viral polymerase complex protein, an M protein, and an NS protein.
55 . The composition of claim 42 , wherein said viral antigen proteins are selected from the group consisting of hemagglutinin and neuraminidase or both.
56 . 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.
57 . The method of claim 56 , wherein the regulatory element for the synthesis of vRNA or cRNA with an exact 3′ end is an RNA polymerase I (pol I) terminator sequence.
58 . The method of claim 56 , wherein the regulatory element for the synthesis of vRNA or cRNA with an exact 3′ end is a ribozyme sequence.
59 . The method of claim 57 , wherein the pol I promoter is proximal to the polyadenylation signal and the pol I terminator sequence is proximal to the pol II promoter.
60 . The method of claim 57 wherein the pol I promoter is proximal to the pol II promoter and the pol I terminator sequence is proximal to the polyadenylation signal.
61 . The method of claim 56 , wherein said negative strand RNA virus virion is an influenza virus.
62 . The method of claim 61 , wherein said influenza virus grows well in culture and pathogenic.
63 . The method of claim 61 , wherein said influenza virus is attenuated and pathogenic.
64 . The method of claim 61 , wherein said influenza virus grows well in culture, is attenuated, and is pathogenic.
65 . The method of claim 63 , wherein said influenza virus is temperature sensitive.
66 . The method of claim 65 , wherein said influenza virus is cold adapted.
67 . The method of claim 62 , wherein said culture is a chicken egg or a host cell.
68 . The method of claim 56 , wherein said viral internal proteins are selected from the group consisting of a viral polymerase complex protein, an M protein, and an NS protein.
69 . The method of claim 56 , wherein said viral antigen proteins are selected from the group consisting of hemagglutinin and neuraminidase or both.
70 . 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.
71 . The method of claim 70 , which further comprises inactivating the virion.
72 . The method of claim 71 , wherein said virion is inactivated by a treatment selected from the group consisting of formaldehyde, beta-propiolactone, ether, ether with a detergent, cetyl trimethyl ammonium bromide (CTAB) and Triton N101, and sodium deoxycholate and tri(n-butyl) phosphate.
73 . The method of claim 70 , which further comprises a clarification of allantoic fluid from virus.
74 . The method of 73 , wherein said virion is inactivated prior to the clarification of allantoic fluid from virus.
75 . The method of claim 73 , wherein the virion is inactivated after the clarification of an allantoic fluid.
76 . The method of claim 70 , which further comprises assessing the potency of said virion by a single radial immunodiffusion (SRI) test.
77 . The method of claim 70 , wherein said vaccine further comprises a carrier.
78 . The method of claim 70 , wherein said vaccine further comprises an adjuvant.
79 . The method of claim 78 , wherein said adjuvant is selected from the group consisting of complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels, aluminum hydroxide, surface active substances, lysolecithin, pluronic polyols, polyanions, peptides, oil, hydrocarbon emulsions, BCG (bacille Calmette-Guerin), Corynebacterium parvum and QS-21.
80 . 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.
81 . The method of claim 80 , wherein said administering is via intramuscular injection.
82 . The method of claim 80 , wherein said administering is via intranasal route.
83 . The method of claim 80 , wherein said subject in need thereof is an animal.
84 . The method of claim 83 , wherein said animal is a human.
85 . The method of claim 80 , wherein said vaccine induces an immune response.
86 . The method of claim 85 , wherein said immune response is a local immunity, a mucosal immunity, a cell mediated immunity, or a humoral immunity, or a combination of one or more immunities thereof.
87 . The method of claim 80 , wherein said vaccinating prevents or treats a disease or disorder associated with a negative strand RNA virus infection when administered to a subject in need thereof.
88 . The method of claim 87 , wherein said administering is via intramuscular injection.
89 . The method of claim 87 , wherein said administering is via intranasal route.
90 . The method of claim 87 , wherein said subject in need thereof is an animal.
91 . The method of claim 90 , wherein said animal is a human.