Abstract
BackgroundOne of the critical tasks in analytical testing is to monitor and assign the infectivity or potency of viral based vaccines from process development to production of final clinical lots. In this study, a high throughput RT-qPCR based approach was developed to evaluate the infectious titre in a replication-defective HSV-2 candidate vaccine, called HSV529. This assay is a combination of viral propagation and quantitative RT-PCR which measures the amount of RNA in infected cells after incubation with test samples.ResultsThe relative infectious titre of HSV529 candidate vaccine was determined by a RT-qPCR method targeting HSV-2 gD2 gene. The data were analyzed using the parallel-line analysis as described in the European Pharmacopoeia 8th edition. The stability of HSV529 test samples were also investigated in a concordance study between RT-qPCR infectivity assay and a classical plaque assays. A suitable correlation was determined between both assays using an identical sample set in both assays. The RT-qPCR infectivity assay was further characterized by evaluating the intermediate precision and accuracy. The coefficient of variation from the six independent assays was less than 10%. The accuracy of each of the assay was also evaluated in the range of 92.91% to 120.57%.ConclusionsOur data demonstrate that the developed RT-qPCR infectivity assay is a rapid high throughput approach to quantify the infectious titer or potency of live attenuated or defective viral-based vaccines, an attribute which is associated with product quality.
Highlights
One of the critical tasks in analytical testing is to monitor and assign the infectivity or potency of viral based vaccines from process development to production of final clinical lots
Specificity of HSV-2 various target genes and optimization of harvest time The accumulation of HSV529 RNA during infection was measured by one step RT-quantitative PCR (qPCR) at 3, 6, 12, 16, and 24 hours post-infection using specific primers for ICP27, TK, and gD2
The overall results show that HSV-2 gD2 is a suitable targeted gene for evaluation of HSV529 infectious titre 12–16 hour post-infection
Summary
One of the critical tasks in analytical testing is to monitor and assign the infectivity or potency of viral based vaccines from process development to production of final clinical lots. A high throughput RT-qPCR based approach was developed to evaluate the infectious titre in a replication-defective HSV-2 candidate vaccine, called HSV529. This assay is a combination of viral propagation and quantitative RT-PCR which measures the amount of RNA in infected cells after incubation with test samples. The conventional in-vitro assays to measure the titer or potency of live viral-based vaccines are usually based on the infectivity of the vaccine virus in cell cultures (plaque assay or CCID50) [1,2,3,4,5] In both methods, the experiment duration is long due to the time needed for virus replication producing the biological effect. A one week time reduction in the qPCR infectivity assay without loss of precision compared to a plaque assay and TCID50 was a major advantage of the assay
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