Abstract
To control circulating vaccine-derived type 2 poliovirus outbreaks, a more genetically stable novel Oral Poliovirus Vaccine type 2 (nOPV2) was developed by targeted modifications of Sabin 2 genome. Since the use of OPV2 made of Sabin 2 strain has been stopped, it is important to exclude the possibility that batches of nOPV2 are contaminated with Sabin 2 virus. Here, we report the development of a simple quantitative one-step reverse-transcription polymerase chain reaction assay for the detection and quantitation of Sabin 2 virus in the presence of overwhelming amounts of nOPV2 strain. The method is specific and linear within 8 log10 range even in the presence of relevant amounts of nOPV2 virus. It is sensitive, with a lower limit of detection of 0.2 CCID50/mL (an equivalent of 198 genome copies per mL), and generates reproducible results. This assay can be used for quality control and lot release of the nOPV2.
Highlights
There are two prophylactic vaccines against poliomyelitis caused by the three serotypes of poliovirus: inactivated poliovirus vaccine (IPV) and live oral poliovirus vaccine (OPV).The use of these vaccines has dramatically reduced polio incidence worldwide
Was serially spiked in nOPV2c1-295 virus (8.07 Log10 CCID50 /mL) suspension as follows: twelve 1.5 mL–tubes were filled with 180 μL of nOPV2c1-295, 20 μL of Sabin 2 virus was added to the first tube containing 180 μL of nOPV2c1-295, pipetted and 20 μL of the mixture transferred to the second tube containing 180 μL of nOPV2c1-295 mixed and 20 μL of the mixture transferred to tube, the same procedure was repeated until the last tube, the last 20 μL of the mixture was discarded, and the RNA was extracted from each dilution and subjected to qosRT-PCR analysis as described above
The results showed that the presence of novel OPV type 2 (nOPV2) virus with high titer did not interfere with the assay that was able to detect Sabin 2 virus at levels of 0.22 CCID50 /mL which correspond to traces of Sabin 2 virus in nOPV2 (2 × 10−7 % of Sabin 2 in nOPV2 suspension) (Tables 3 and 4)
Summary
There are two prophylactic vaccines against poliomyelitis caused by the three serotypes of poliovirus: inactivated poliovirus vaccine (IPV) and live oral poliovirus vaccine (OPV). The Global Polio Eradication Initiative (GPEI) led to the eradication of wild polioviruses of types 2 and 3 [5], but wild type-1 poliovirus remains endemic in Afghanistan and Pakistan [6] This progress was achieved mostly because of the massive use of OPV that stimulates strong systemic and mucosal immunity [7]. To overcome the limitations of current vaccines, two genetically-stabilized novel OPV type 2 (nOPV2) vaccine candidates were developed [18,19] Both candidates include an alternative domain V from the S15 strain [19] which incorporated 18 nucleotide substitutions (compared to Sabin 2 strain) aimed at avoiding thermodynamic stabilization of the structure that leads to the increase of neurovirulence. The assay for the presence of Sabin 2 contamination in nOPV2 stocks is simple, rapid, reproducible, sensitive, and has large linearity range suitable for quality control of the nOPV2 vaccine
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