Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852, USA.
Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):20063-8. doi: 10.1073/pnas.1012537107. Epub 2010 Nov 1.
Intrinsic genetic instability of RNA viruses may lead to the accumulation of revertants during manufacture of live viral vaccines, requiring rigorous quality control to ensure vaccine safety. Each lot of oral poliovirus vaccine (OPV) is tested for neurovirulence in animals and also for the presence of neurovirulent revertants. Mutant analysis by PCR and restriction enzyme cleavage (MAPREC) is used to measure the frequency of neurovirulent mutations at the 5' untranslated region (UTR) of the viral genome that correlate with the level of neurovirulence determined by the monkey neurovirulence test. However, MAPREC can only monitor mutations at a few genomic loci and miss mutations at other sites that could adversely affect vaccine quality. Here we propose to use massively parallel sequencing (MPS) for sensitive detection and quantification of all mutations in the entire genome of attenuated viruses. Analysis of vaccine samples and reference preparations demonstrated a perfect agreement with MAPREC results. Quantitative MPS analysis of validated reference preparations tested by MAPREC produced identical results, suggesting that the method could take advantage of the existing reference materials and be used as a replacement for the MAPREC procedure in lot release of OPV. Patterns of mutations present at a low level in vaccine preparations were characteristic of seed viruses used for their manufacture and could be used for identification of individual batches. This approach may represent the ultimate tool for monitoring genetic consistency of live viral vaccines.
RNA 病毒固有的遗传不稳定性可能导致活病毒疫苗生产过程中回复突变体的积累，因此需要严格的质量控制来确保疫苗的安全性。每批口服脊髓灰质炎病毒疫苗（OPV）都要在动物身上进行神经毒性测试，并检测是否存在神经毒性回复突变体。通过聚合酶链反应和限制性内切酶切割（MAPREC）进行的突变分析用于测量与猴神经毒性试验确定的神经毒性水平相关的病毒基因组 5'非翻译区（UTR）中神经毒性突变的频率。然而，MAPREC 只能监测少数基因组位点的突变，而错过其他可能对疫苗质量产生不利影响的位点的突变。在这里，我们建议使用大规模平行测序（MPS）对减毒病毒的整个基因组中的所有突变进行敏感检测和定量。对疫苗样本和参考制剂的分析与 MAPREC 结果完全一致。通过 MAPREC 测试的经验证的参考制剂的定量 MPS 分析产生了相同的结果，这表明该方法可以利用现有的参考材料，并作为 OPV 批次放行中 MAPREC 程序的替代方法。在疫苗制剂中以低水平存在的突变模式是其制造所用种子病毒的特征，可用于鉴定各个批次。这种方法可能代表了监测活病毒疫苗遗传一致性的最终工具。
