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.

Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes

“…This virus-serum mixture was then added onto Vero cells in 96-well plates and incubated for 4 days. Serum end-point neutralization titers (LiCor 50 ) were defined as the reciprocal of the highest serum dilution that blocks 50% of the DENV infection when compared to virus control included on each assay plate, using an infrared Odyssey Sa imaging system (Li-Cor Biosciences) [81].…”

A Role for Human Skin Mast Cells in Dengue Virus Infection and Systemic Spread

“…In addition, there have been recent outbreaks in Texas and Florida where transmission occurred on American soil (9, 10). There are no vaccines or specific therapeutic agents approved for DENV, aside from the Sanofi Pasteur vaccine (Dengvaxia) that is recommended by the World Health Organization for limited use in highly endemic regions (11). The development of a safe and effective vaccine has been hindered by the phenomenon of Ab-dependent enhancement, where Abs against one serotype can lead to severe disease, such as dengue hemorrhagic fever and dengue shock syndrome, upon infection with a second serotype (8,12,13).…”

Designing a polytope for use in a broad-spectrum dengue virus vaccine

“…In 2015, the World Health Organization (WHO) approved a new dengue vaccine, CYD-TDV, and several additional candidate vaccines are currently undergoing clinical development 9 . Several of these vaccines, including CYD-TDV, are live attenuated vaccines 10, 11, 12. These live attenuated vaccines have associated risks because pathogens are used as the vaccinating agent.…”