title: A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.
abstract: Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.
authors.affiliation: Merck Research Laboratories, Merck & Co., Kenilworth, NJ, United States of America.
authors.forename: Dhanasekaran
authors.initials: D
authors.lastname: Govindarajan
authors.affiliation: Merck Research Laboratories, Merck & Co., Kenilworth, NJ, United States of America.
authors.forename: Liming
authors.initials: L
authors.lastname: Guan
authors.affiliation: Merck Research Laboratories, Merck & Co., Kenilworth, NJ, United States of America.
authors.forename: Steven
authors.initials: S
authors.lastname: Meschino
authors.affiliation: Merck Research Laboratories, Merck & Co., Kenilworth, NJ, United States of America.
authors.forename: Arthur
authors.initials: A
authors.lastname: Fridman
authors.affiliation: Merck Research Laboratories, Merck & Co., Kenilworth, NJ, United States of America.
authors.forename: Ansu
authors.initials: A
authors.lastname: Bagchi
authors.affiliation: Merck Research Laboratories, Merck & Co., Kenilworth, NJ, United States of America.
authors.forename: Irene
authors.initials: I
authors.lastname: Pak
authors.affiliation: Immune Design Corporation, Seattle, WA, United States of America.
authors.forename: Jan
authors.initials: J
authors.lastname: ter Meulen
authors.affiliation: Merck Research Laboratories, Merck & Co., Kenilworth, NJ, United States of America.
authors.forename: Danilo R
authors.initials: DR
authors.lastname: Casimiro
authors.affiliation: Merck Research Laboratories, Merck & Co., Kenilworth, NJ, United States of America.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bett
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003715:Dengue / Q000517:prevention & control; D053059:Dengue Vaccines / Q000138:chemical synthesis* / Q000276:immunology; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology*; D005260:Female; D008253:Macaca mulatta; D008297:Male; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics; D019444:Vaccines, DNA / Q000138:chemical synthesis* / Q000276:immunology; D014709:Vero Cells / Q000821:virology; D015005:Yellow fever virus / Q000235:genetics
chemical_list: D053059:Dengue Vaccines; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA