Arboviral Diseases Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA.
PLoS Negl Trop Dis. 2013 May 30;7(5):e2243. doi: 10.1371/journal.pntd.0002243. Print 2013.
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.
我们开发了一种制造策略，可以提高重组减毒嵌合登革热疫苗（DENVax）病毒的安全性和遗传稳定性。这些病毒在减毒登革热 2 型 PDK-53 候选疫苗病毒的复制背景中包含登革热血清型 1-4 的前膜（prM）和包膜（E）基因，是在 cGMP 下制造的。
方法/主要发现：从 RNA 转染的 Vero 细胞中衍生出疫苗病毒后，每种血清型生产 6 个噬菌斑纯化病毒。然后分析纯化的噬菌斑以选择一个用于生成主种子的库存。对主病毒种子进行了全面的遗传和表型特征分析，以确保这些病毒保留了疫苗病毒先前确定的减毒决定因素和表型。我们还评估了疫苗病毒在同域（泰国）埃及伊蚊媒介中的载体能力。
结论/意义：所有四种血清型的主疫苗种子都保留了先前定义的安全特征，包括三个主要的减毒遗传位点、小斑、哺乳动物细胞中的温度敏感性、在蚊细胞培养物中的复制减少以及在新生小鼠中的神经毒力降低。此外，候选疫苗病毒在埃及伊蚊中表现出大大降低的感染和传播能力，并且不太可能由这些蚊子传播。该制造策略已成功用于生产候选四价疫苗，目前正在美国、中美洲和南美洲以及亚洲进行人体临床试验。
