PDF(Pubmed)
Abstract：
Recently, a multiplex PCR-based titration (MPBT) assay was developed for simultaneous determination of infectious titers of all three Sabin strains of the oral poliovirus vaccine (OPV) to replace the conventional CCID50 assay, which is both time-consuming and laborious. The MPBT assay was shown to be reproducible, robust and sensitive. The conventional and MPBT assays showed similar results and sensitivity. The MPBT assay can be completed in two to three days, instead of ten days for the conventional assay. To prevent attenuated vaccine strains of poliovirus from reversion to virulence, a novel, genetically stable OPV (nOPV) was developed by modifying the genomes of conventional Sabin strains used in OPV. In this work, we evaluated the MPBT assay as a rapid screening tool to support trivalent nOPV (tnOPV) formulation development by simultaneous titration of the three nOPV strains to confirm stability as needed, for the selection of the lead tnOPV formulation candidate. We first assessed the ability of the MPBT assay to discriminate a 0.5 log10 titer difference by titrating the two tnOPV samples (undiluted and threefold-diluted) on the same plate. Once the assay was shown to be discriminating, we then tested different formulations of tnOPV drug products (DPs) that were subjected to different exposure times at 37 °C (untreated group and treated groups: 2 and 7 days at 37 °C), and to three freeze and thaw (FT) cycles. Final confirmation of the down selected formulation candidates was achieved by performing the conventional CCID50 assay, comparing the stability of untreated and treated groups and FT stability testing on the top three candidates. The results showed that the MPBT assay generates similar titers as the conventional assay. By testing two trivalent samples in the same plate, the assay can differentiate a 0.5 log10 difference between the titers of the tested nOPV samples. Also, the assay was able to detect the gradual degradation of nOPV viruses with different formulation compositions and under different time/temperature conditions and freeze/thaw cycles. We found that there were three tnOPV formulations which met the stability criteria of less than 0.5 log10 loss after 2 days\' exposure to 37 ℃ and after three FT cycles, maintaining the potency of all three serotypes in these formulations. The ability of the MPBT assay to titrate two tnOPV lots (six viruses) in the same plate makes it cheaper and gives it a higher throughput for rapid screening. The assay detected the gradual degradation of the tnOPV and was successful in the selection of optimal formulations for the tnOPV. The results demonstrated that the MPBT method can be used as a stability indicating assay to assess the thermal stability of the nOPV. It can be used for rapid virus titer determination during the vaccine manufacturing process, and in clinical trials. The MPBT assay can be automated and applied for other viruses, including those with no cytopathic effect.
摘要：
最近,开发了一种基于多重PCR的滴定（MPBT）测定法，用于同时测定口服脊髓灰质炎病毒疫苗（OPV）的所有三种萨宾菌株的感染滴度，以取代常规的CCID50测定法，这既费时费力。MPBT分析被证明是可重复的，健壮和敏感。常规和MPBT测定显示相似的结果和灵敏度。MPBT检测可以在两到三天内完成，而不是常规检测的十天。为了防止脊髓灰质炎病毒减毒疫苗株逆转为毒力，一本小说,遗传稳定的OPV（nOPV）是通过修饰OPV中使用的常规Sabin菌株的基因组而开发的。在这项工作中,我们评估了MPBT测定作为一种快速筛选工具，通过同时滴定三种nOPV菌株来支持三价nOPV（tnOPV）制剂开发，以确认所需的稳定性，用于选择主要的tnOPV配方候选。我们首先通过在同一板上滴定两个tnOPV样品(未稀释和三倍稀释)来评估MPBT测定区分0.5log10滴度差异的能力。一旦分析被证明是有区别的，然后，我们测试了在37°C下经历不同暴露时间的tnOPV药物产品（DP）的不同配方（未处理组和处理组：在37°C下2天和7天），和三个冻融(FT)循环。通过进行常规CCID50测定，最终确认了向下选择的候选制剂。比较未治疗组和治疗组的稳定性，并对前三名候选人进行FT稳定性测试。结果显示MPBT测定产生与常规测定相似的滴度。通过在同一板上测试两个三价样品，该测定可以区分测试的nOPV样品的滴度之间的0.5log10差异。此外，该测定能够检测具有不同制剂组成和在不同时间/温度条件和冷冻/解冻循环下的nOPV病毒的逐渐降解。我们发现，有三种tnOPV制剂在暴露于37℃2天后和三个FT循环后，满足小于0.5log10损失的稳定性标准，维持这些制剂中所有三种血清型的效力。MPBT测定在同一平板中滴定两个tnOPV批次（六个病毒）的能力使其更便宜，并为快速筛选提供了更高的通量。该测定检测到tnOPV的逐渐降解,并且成功地选择了tnOPV的最佳制剂。结果表明，MPBT方法可用作稳定性指示测定法，以评估nOPV的热稳定性。可用于疫苗生产过程中病毒滴度的快速测定,在临床试验中。MPBT测定可以自动化并应用于其他病毒，包括那些没有细胞病变效应的。
