Genetic and phenotypic characterization of manufacturing seeds for a tetravalent dengue vaccine (DENVax)

Abstract

Background: We 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 findings: After 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/significance: All 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.

Figure 1. Plaque sizes of the DENVax MVS.

Mean plaque diameters (mm) ± SD (error bars) of the virus plaques in Vero cells under agarose overlay measured on day 9 pi. The wt DENVs and previously published research-grade vaccine candidate viruses were included for control and comparison.

Figure 2. Temperature sensitivities of DENVax MVS.

Mean titers ± SD (error bars) of the viruses replicated in Vero cells at 37°C or 39°C. The wt DENVs and previously published research-grade vaccine candidate viruses were included for comparison. The DENVax-4 MVS contains additional F-127 that can mask the temperature sensitivity results of the virus in this assay. A separate experiment analyzing a surrogate DENVax-4 in the absence of F127 was also included.

Figure 3. Restricted growth of DENVax MVS in C6/36 cells.

Mean titers ± SD (error bars) of the viruses replicated in C6/36 cells 6 days pi. The wt DENVs and previously published research-grade vaccine candidate viruses were included for comparison.

Figure 4. Neurovirulence in newborn mice.

Pooled results of numerous experiments summarizing the neurovirulence of wt DENV-2 16681 virus in CDC-ICR (n = 72) and Taconic-ICR (n = 32) newborn mice challenged ic with 10⁴ pfu of the virus (A). Neurovirulence of DENVax MVS tested in Taconic-ICR mice with a dose of 10⁴ pfu (B) or 10³ pfu (C). The numbers of animals tested per group in one experiment (n = 16) or two pooled experiments (n = 31 or 32) are indicated.