Genetic and Phenotypic Characterization of Manufacturing Seeds for a Tetravalent Dengue Vaccine (DENVax)

Abstract

BackgroundWe have developed a manufacturing strategy that can improve the safety and genetic stability of recombinant live-attenuated chimeric dengue vaccine (DENVax) viruses. These viruses, containing the pre-membrane (prM) and envelope (E) genes of dengue serotypes 1–4 in the replicative background of the attenuated dengue-2 PDK-53 vaccine virus candidate, were manufactured under cGMP.Methodology/Principal FindingsAfter deriving vaccine viruses from RNA-transfected Vero cells, six plaque-purified viruses for each serotype were produced. The plaque-purified strains were then analyzed to select one stock for generation of the master seed. Full genetic and phenotypic characterizations of the master virus seeds were conducted to ensure these viruses retained the previously identified attenuating determinants and phenotypes of the vaccine viruses. We also assessed vector competence of the vaccine viruses in sympatric (Thai) Aedes aegypti mosquito vectors.Conclusion/SignificanceAll four serotypes of master vaccine seeds retained the previously defined safety features, including all three major genetic loci of attenuation, small plaques, temperature sensitivity in mammalian cells, reduced replication in mosquito cell cultures, and reduced neurovirulence in new-born mice. In addition, the candidate vaccine viruses demonstrated greatly reduced infection and dissemination in Aedes aegypti mosquitoes, and are not likely to be transmissible by these mosquitoes. This manufacturing strategy has successfully been used to produce the candidate tetravalent vaccine, which is currently being tested in human clinical trials in the United States, Central and South America, and Asia.

Dengue Virus Entry and Trafficking: Perspectives as Antiviral Target for Prevention and Therapy

“…In Vero cells, a cell line usually employed for DENV quantification and for the development and production of DENV vaccines [80][81][82], alternative internalization routes can be used depending on the virus serotype. By treatment with biochemical inhibitors and overexpression of cellular proteins involved in the different endocytic routes, it was proved that the entry of DENV-1 for a productive infection requires clathrin-mediated endocytosis, whereas the infectious entry of DENV-2 into Vero cells occurs by a nonclassical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin [74].…”

Trials and tribulations on the path to developing a dengue vaccine

“…Takeda Pharmaceuticals is developing a tetravalent chimeric dengue vaccine candidate by introducing DENV-1, -3, and -4 prM and E genes into cDNA derived from a live attenuated DENV-2 component [44][45][46][47][48][49][50][51][52]. A Phase 1 dose-escalation trial in DENV-nonimmune subjects, involving a single dose of vaccine formulations with low or high DENV viral concentrations administered SQ or intradermally (ID), demonstrated acceptable safety.…”

Vaccine Development as a Means to Control Dengue Virus Pathogenesis: Do We Know Enough?

“…The principal attenuating mutations in the DENV-2 backbone common to each component of DENVax include mutations in position 57 of the 5 UTR, residue 53 of NS1, and residue 250 of NS3 (58). Analysis of the genetic stability of the four components of DENVax during the preparation of master viral seed stocks revealed that the two attenuating mutations in the nonstructural proteins were stable: Low levels of reversion were noted with the UTR mutation (59). Clinical studies of two-dose regiments of DENVax are underway in the United States, Central and South America, and Asia.…”