Manukyan Hasmik, Tritama Erman, Wahid Rahnuma, Anstadt Jennifer, Konz John, Chumakov Konstantin, Laassri Majid
Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA.
Research and Development Division, PT. BioFarma, Bandung 40161, Indonesia.
Vaccines (Basel). 2023 Nov 19;11(11):1729. doi: 10.3390/vaccines11111729.
Recently, genetically stable novel OPVs (nOPV) were developed by modifying the genomes of Sabin viruses of conventional OPVs to reduce the risk of reversion to neurovirulence and therefore the risk of generating circulating vaccine-derived polioviruses. There is a need for specific and sensitive methods for the identification and quantification of nOPV viruses individually and in mixtures for clinical trials and potentially for manufacturing quality control and environmental surveillance. In this communication, we evaluated and improved the quantitative multiplex one-step reverse transcriptase polymerase chain reaction (qmosRT-PCR) assay for the identification and quantification of nOPV viruses in samples with different formulations and virus concentrations and in virus-spiked stool samples. The assay was able to specifically identify at least 1 log CCID/mL of each serotype in the presence of the two other serotypes at high concentrations (6-7 log CCID/mL) in the same sample. In addition, the lowest viral concentration that the assay was able to detect in stool samples was 17 CCID/mL for nOPV1 and nOPV2 viruses and 6 CCID/mL for nOPV3. We also found high correlation between the expected and observed (by qmosRT-PCR) concentrations of spiked viruses in stool samples for all three nOPV viruses, with R-squared values above 0.95. The analysis of samples collected from an nOPV2 clinical trial showed that 100% of poliovirus type 2 was detected and few samples showed the presence of type 1 and 3 residuals from previous vaccinations with bOPV (at least 4 weeks prior vaccination with nOPV2), confirming the high sensitivity of the method. The qmosRT-PCR was specific and sensitive for the simultaneous identification and quantification of all three nOPV viruses. It can be used as an identity test during the nOPV manufacturing process and in evaluation of virus excretion in nOPV clinical trials.
最近，通过对传统口服脊髓灰质炎疫苗（OPV）的萨宾病毒基因组进行改造，开发出了基因稳定的新型OPV（nOPV），以降低恢复为神经毒力的风险，从而降低产生循环疫苗衍生脊髓灰质炎病毒的风险。需要有特异性和灵敏性的方法来单独和混合鉴定及定量nOPV病毒，用于临床试验，并可能用于生产质量控制和环境监测。在本通讯中，我们评估并改进了定量多重一步逆转录聚合酶链反应（qmosRT-PCR）检测方法，用于鉴定和定量不同配方和病毒浓度的样品以及病毒加标的粪便样品中的nOPV病毒。该检测方法能够在同一样品中存在高浓度（6-7 log CCID/mL）的其他两种血清型的情况下，特异性地鉴定出每种血清型至少1 log CCID/mL的病毒。此外，该检测方法在粪便样品中能够检测到的最低病毒浓度，对于nOPV1和nOPV2病毒为17 CCID/mL，对于nOPV3为6 CCID/mL。我们还发现，对于所有三种nOPV病毒，粪便样品中加标病毒的预期浓度与（通过qmosRT-PCR）观察到的浓度之间具有高度相关性，决定系数（R平方）值高于0.95。对一项nOPV2临床试验收集的样品进行分析表明，100%检测到了2型脊髓灰质炎病毒，少数样品显示存在先前接种bOPV（在接种nOPV2前至少4周）后残留的1型和3型病毒，证实了该方法的高灵敏度。qmosRT-PCR对于同时鉴定和定量所有三种nOPV病毒具有特异性和灵敏性。它可用于nOPV生产过程中的鉴别试验以及评估nOPV临床试验中的病毒排泄情况。
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