Engineered respiratory syncytial viruses with control of cell-to-cell virus transmission for enhanced safety of live virus vaccines

Abstract

Highly antigenic yet safe vaccines against diseases caused by Paramyxoviridae viruses such as respiratory syncytial virus (RSV) are provided. The vaccines comprise attenuated Paramyxoviridae viruses with high antigenicity but which display impaired cell-to-cell transmission as a result of genetic manipulation of the gene encoding the matrix (M) protein. In the viruses, the M protein is absent or mutated to a less active form. Screening or assay systems and methods for evaluating the infectivity of mutant M proteins and for identifying suitable M candidates for live-attenuated vaccine virus and VLP production, are also provided.

We claim: 
     
         1 . A recombinant, live attenuated virus in which a gene encoding a matrix (M) protein is
 i) deleted;   ii) genetically manipulated to produce an M protein with no or decreased activity; or   iii) genetically manipulated to produce a decreased amount of said M protein.   
     
     
         2 . The recombinant, live attenuated virus of  claim 1 , wherein the recombinant, live attenuated virus is a  Paramyxoviridae  virus. 
     
     
         3 . The recombinant, live attenuated virus of  claim 1 , wherein the recombinant, live attenuated virus is a member of the group consisting of RNA viruses,  Mononegavirales  order viruses,  Paramyxoviridae  viruses and retroviruses. 
     
     
         4 . The recombinant, live attenuated virus of  claim 2 , wherein the  Paramyxoviridae  virus is a  Pneumovirus  virus. 
     
     
         5 . The recombinant, live attenuated virus of  claim 2 , wherein the  Pneumovirus  virus is a respiratory syncytial virus (RSV). 
     
     
         6 . The recombinant, live attenuated virus of  claim 5 , wherein the RSV is human RSV. 
     
     
         7 . A pharmaceutical composition comprising the recombinant, live, attenuated virus of  claim 1  in an amount sufficient to elicit an immune response in a host. 
     
     
         8 . A method of immunizing a subject against symptoms of disease caused by a  Paramyxoviridae  virus, comprising the step of administering to said subject at least one dose of the pharmaceutical composition of  claim 6 . 
     
     
         9 . The method of  claim 8 , wherein said  Paramyxoviridae  virus is RSV and said subject is selected from the group consisting of a child, an immunocompromised individual, and an elderly individual. 
     
     
         10 . A viral replication assessment system, comprising
 a host cell comprising nucleic acid sequences comprising a transcriptional control element operably linked to nucleic acid sequences encoding a mutant M protein; and   an M-null virus.   
     
     
         11 . The system of  claim 10 , wherein said transcriptional control element is a constitutive promoter. 
     
     
         12 . The system of  claim 11 , wherein said consitutive promoter is a cytomegalovirus (CMV) promoter. 
     
     
         13 . The system of  claim 10 , wherein said transcriptional control element is inducible and said M-null virus comprises nucleic acid sequences encoding at least one gene product capable of activating said inducible transcriptional control element. 
     
     
         14 . The system of  claim 13 , wherein said inducible transcriptional control element comprises tetracycline response elements (TRE) and said at least one gene product is tetracycline transactivating protein (Tet). 
     
     
         15 . The system of  claim 13 , wherein said nucleic acid sequences comprising an inducible transcriptional control element are present at a location selected from the group consisting of: within a virus; on a plasmid; on a mini-replicon; and within the host cell genome. 
     
     
         16 . The system of  claim 10 , wherein said virus is a  Paramyxoviridae  virus. 
     
     
         17 . The system of  claim 10 , wherein said mutant M protein is a double alanine mutant M protein. 
     
     
         18 . A method of assessing viral replication, comprising the steps of providing a plurality of host cells, each of which comprises nucleic acid sequences comprising a transcriptional control element operably linked to nucleic acid sequences encoding said mutant M protein;
 infecting at least one of said plurality of host cells with at least one M-null virus; and measuring a titer of viral progeny produced in said plurality of host cells.   
     
     
         19 . The method of  claim 18 , wherein said transcriptional control element is a consitutive promoter. 
     
     
         20 . The method of  claim 19 , wherein said consitutive promoter is a cytomegolovirus (CMV) promoter. 
     
     
         21 . The method of  claim 17 , wherein said transcriptional control element is inducible and said M-null virus comprises nucleic acid sequences encoding at least one gene product capable of activating said inducible transcriptional control element. 
     
     
         22 . The system of  claim 21 , wherein said inducible transcriptional control element comprises tetracycline response elements (TRE) and said at least one gene product is tetracycline transactivating protein (Tet). 
     
     
         23 . The system of  claim 21 , wherein said nucleic acid sequences comprising an inducible transcriptional control element is present at a location selected from the group consisting of: within a virus; on a plasmid; on a mini-replicon; and within the host cell genome. 
     
     
         24 . The system of  claim 18 , wherein said virus is a  Paramyxoviridae  virus. 
     
     
         25 . The system of  claim 18 , wherein said mutant M protein is a double alanine mutant M protein. 
     
     
         26 . A method of identifying M protein mutations that alter infectious virus production, comprising the steps of
 providing a plurality of host cells, each of which comprises nucleic acid sequences comprising a transcriptional control element operably linked to nucleic acid sequences encoding an M protein with a mutation;   infecting at least one of said plurality of host cells with at least one M-null virus;   measuring a titer of viral progeny produced in said plurality of host cells; and if said titer of viral progeny differs from a control titer of viral progeny obtained with wild type virus, then identifying said mutation in said M protein as an M protein mutation that alters infectious virus production.   
     
     
         27 . The method of  claim 26 , wherein when said titer of viral progeny is less than said control titer of viral progeny obtained with wild type virus, then said mutation in said M protein is identified as an M protein mutation that decreases infectious virus production. 
     
     
         28 . The method of  claim 26 , wherein when said titer of viral progeny is greater than said control titer of viral progeny obtained with wild type virus, then said mutation in said M protein is identified as an M protein mutation that increases infectious virus and VLP production. 
     
     
         29 . A method of making a virus with a decreased capacity for virus production compared to a wild type control virus, comprising the steps of providing a plurality of host cells, each of which comprises nucleic acid sequences comprising a transcriptional control element operably linked to nucleic acid sequences encoding an M protein with a mutation;
 infecting at least one of said plurality of host cells with at least one M-null virus;   measuring a titer of viral progeny produced in said plurality of host cells; and   if said titer of viral progeny is less than a control titer of viral progeny obtained with wild type virus, then identifying said mutation in said M protein as a mutation that decreases infectious virus production; and   genetically engineering a virus to contain and express nucleic acid sequences encoding said M protein with said mutation.