Dengue type 4 live-attenuated vaccine viruses passaged in Vero cells affect genetic stability and dengue-induced hemorrhaging in mice
Journal
PLoS ONE
Journal Volume
6
Journal Issue
10
Date Issued
2011
Author(s)
Abstract
Most live-attenuated tetravalent dengue virus vaccines in current clinical trials are produced from Vero cells. In a previous study we demonstrated that an infectious cDNA clone-derived dengue type 4 (DEN-4) virus retains higher genetic stability in MRC-5 cells than in Vero cells. For this study we investigated two DEN-4 viruses: the infectious cDNA clone-derived DEN-4 2A and its derived 3′ NCR 30-nucleotide deletion mutant DEN-4 2AΔ30, a vaccine candidate. Mutations in the C-prM-E, NS2B-NS3, and NS4B-NS5 regions of the DEN genome were sequenced and compared following cell passages in Vero and MRC-5 cells. Our results indicate stronger genetic stability in both viruses following MRC-5 cell passages, leading to significantly lower RNA polymerase error rates when the DEN-4 virus is used for genome replication. Although no significant increases in virus titers were observed following cell passages, DEN-4 2A and DEN-4 2AΔ30 virus titers following Vero cell passages were 17-fold to 25-fold higher than titers following MRC-5 cell passages. Neurovirulence for DEN-4 2A and DEN-4 2AΔ30 viruses increased significantly following passages in Vero cells compared to passages in MRC-5 cells. In addition, more severe DEN-induced hemorrhaging in mice was noted following DEN-4 2A and DEN-4 2AΔ30 passages in Vero cells compared to passages in MRC-5 cells. Target mutagenesis performed on the DEN-4 2A infectious clone indicated that single point mutation of E-Q 438H, E-V 463L, NS2B-Q 78H, and NS2B-A 113T imperatively increased mouse hemorrhaging severity. The relationship between amino acid mutations acquired during Vero cell passage and enhanced DEN-induced hemorrhages in mice may be important for understanding DHF pathogenesis, as well as for the development of live-attenuated dengue vaccines. Taken together, the genetic stability, virus yield, and DEN-induced hemorrhaging all require further investigation in the context of live-attenuated DEN vaccine development. ? 2011 Lee et al.
SDGs
Other Subjects
complementary DNA; dengue vaccine; live vaccine; RNA polymerase; amino acid; live vaccine; mutant protein; nucleotide; virus protein; animal experiment; article; bleeding; cell strain; cell strain mrc 5; controlled study; dengue; Dengue virus 4; disease severity; error; gene deletion; gene replication; gene sequence; genetic stability; human; human cell; mouse; newborn; nonhuman; point mutation; Vero cell; virus genome; virus load; virus mutant; virus strain; virus virulence; animal; C57BL mouse; Cercopithecus; dengue; Dengue virus; genetics; immunocompetence; immunology; metabolism; mutation; pathogenicity; Vero cell; virology; virulence; virus culture; virus gene; Dengue virus; Mus; Amino Acids; Animals; Animals, Newborn; Cercopithecus aethiops; Dengue Hemorrhagic Fever; Dengue Virus; Genes, Viral; Hemorrhage; Humans; Immunocompetence; Mice; Mice, Inbred C57BL; Mutant Proteins; Mutation; Nucleotides; Serial Passage; Vaccines, Attenuated; Vero Cells; Viral Nonstructural Proteins; Virulence
Type
journal article