Rapid method of generating live attenuated vaccines

Abstract

The present invention relates to a method of generating a live attenuated vaccine. The present invention also relates to a live attenuated vaccine produced according to the method of the invention.

1 . A method of generating a live attenuated vaccine (LAV) comprising the steps of:
 a) modifying an original virus to generate at least one genetically distinct maladapted virus;   b) infecting a host cell with said at least one maladapted virus;   c) selecting a host cell that displays a preselected phenotype in response to said infection with said at least one maladapted virus and isolating the viral nucleic acid of said maladapted virus from the host cell;   d) sequencing the isolated viral nucleic acid of said maladapted virus and comparing this to the nucleic acid sequence of the original virus;   e) reconstructing the maladapted virus from the original virus to produce a candidate live attenuated vaccine; and   f) screening said candidate live attenuated vaccine for a predetermined phenotype.   
     
     
         2 . The method of  claim 1 , wherein the modification of a virus in step a) is by passaging the virus at least once in a cell that is of a species distinct from the intended recipient of the LAV, or by exposure to a chemical compound, or by culture at a temperature lower than 37° C., or by random mutagenesis, or a combination thereof. 
     
     
         3 . The method of  claim 2 , wherein the chemical compound is ribavirin or 5-fluorouracil (5-FU). 
     
     
         4 . The method of  claim 2 , wherein the random mutagenesis is error prone polymerase chain reaction (PCR). 
     
     
         5 . The method of  claim 1 , wherein the original virus to be modified in step a) is derived from a clinical isolate. 
     
     
         6 . The method of  claim 1 , wherein the host cell of step b) is a cell of human origin. 
     
     
         7 . The method of  claim 6 , wherein the host cell is HuH-7 or HEK293T. 
     
     
         8 . The method of  claim 6 , wherein the host cell comprises an inducible reporter gene operably linked to at least one promoter associated with innate immunity. 
     
     
         9 . The method of  claim 8 , wherein the inducible reporter comprises an ISRE operably linked to a green fluorescent protein (GFP) gene. 
     
     
         10 . The method of  claim 9 , wherein the ISRE is associated with an IRF3-mediated response. 
     
     
         11 . The method of  claim 1 , wherein the preselected phenotype in step c) is plaque size in a plaque assay or expression of a reporter gene. 
     
     
         12 . The method of  claim 11 , wherein the preselected phenotype is small plaque size relative to a host cell that has been infected with the original virus. 
     
     
         13 . The method of  claim 11 , wherein the selection of the host cell that expresses the reporter gene in step c) is by fluorescence-activated cell sorting (FACS) or magnetic activated cell sorting (MACS) or plaque pickup. 
     
     
         14 . The method of  claim 1 , wherein sequencing of the isolated viral nucleic acid in step d) is by Sanger sequencing or Next-Generation Sequencing. 
     
     
         15 . The method of  claim 1 , wherein reconstruction of the maladapted virus in step e) comprises performing site directed mutagenesis on the nucleic acid sequence of the original virus to conform said original viral nucleic acid sequence to the nucleic acid sequence of the maladapted virus. 
     
     
         16 . The method of  claim 1 , wherein screening in step f) is performed in vitro and/or in vivo. 
     
     
         17 . The method of  claim 1 , wherein the predetermined phenotype of step f) comprises one or more of increased replication rate, immunogenicity, generation of small plaques in plaque assays and increased rate of virus uptake, decreased viremia levels in an animal model, increased survival rate in an animal model, decreased weight loss in an animal model, sterilizing immunity rate relative to at least one other candidate vaccine. 
     
     
         18 . The method of  claim 1 , wherein the virus is selected from the group consisting of dengue virus, yellow fever virus, respiratory syncytial virus, cytomegalovirus, Kaposi's sarcoma-associated herpes virus, Epstein-Barr virus, Human papillomavirus, Japanese encephalitis virus, human immunodeficiency virus (HIV) and Zika virus. 
     
     
         19 . The method of  claim 1 , wherein the maladapted virus sequence is mutated at one or more positions in the maladapted virus sequence with respect to the nucleic acid sequence of the original virus. 
     
     
         20 . The method of  claim 1 , wherein the maladapted virus is mutated at 876T>C, or 2925A>G, or both. 
     
     
         21 . A live attenuated vaccine produced according to the method of  claim 1 .