There are several challenges with conventional in-vitro assays (plaque or CPE) to measure the titer of live attenuated or defective viral-based vaccines
[4, 6]. The traditional assays are usually laborious and sometimes hard to interpret. The time due to-assay-completion and cell substrate limitations are also challenges with the conventional in-vitro assays. For instance, it takes nine days to measure the infectious titre in measles or rubella vaccines
. Furthermore, traditional methods require virus neutralization for characterization of infectivity or potentially potency in multivalent viral vaccines. However, a PCR-infectivity based approach does not require virus neutralization, making it a more attractive alternative for multivalent viral vaccines. Although HSV529 candidate vaccine has not been faced with some of these challenges (the HSV-2 virus is able to form plaques in AV529-19 cells over 3 days and is not a multivalent vaccine), a RT-qPCR infectivity based-approach was developed to enhance the assay’s throughput (testing more samples in a shorter time).
During HSV-2 replication, the five viral genes expressed in the immediate-early (phase α), encode regulatory proteins
[10, 11]. After the immediate-early step, early genes are activated (phase β), and these encode proteins required for replication of the viral genome. After genome replication in the early phase, the late step (phase γ) occurs, where HSV-2 structural proteins are expressed and the virus is formed
[10, 11]. One of the critical features of the RT-qPCR infectivity assay was to determine the specificity of the assay targeting appropriate HSV-2 gene. Therefore, one gene (ICP27, TK, and gD2) from each of the replication phases was targeted. We were able to observe a linear relationship between the logarithm of the HSV529 concentration and the C
values by targeting the gD2 gene and not the ICP27 or TK genes. It has to be noted that during the late gD2 expression, the immediate-early and early proteins are also generated and the full form of the virus is completed. HSV-2 gD2 RNA accumulation starts to level off approximately 12 hours post-infection and remains relatively steady for up to 16 hour post-infection.
The developed assay is a combination of in-vitro HSV529 propagation in the suitable cell line for a short HSV-2 replication cycle followed by a RT-qPCR. The infectious titers of the test samples are estimated relative to an in-house reference control. This in-house reference control was titrated in the lab using conventional plaque assay and validated based on 30 independent assays accordance to the International Conference on Harmonisation (ICH) guideline
. Therefore, the assay measures the relative infectious unit based on the in-house reference control unitage. Briefly, confluent AV529 cells in 96-well plates were inoculated with serial dilutions of HSV529 test samples and an HSV529 in-house control, to produce a standard curve followed by incubation for 16 hours. Thereafter, HSV529-19 cells were lysed, total RNA purified, DNAse treated, and RT-qPCR targeting HSV-2 gD2 gene was performed to quantitate HSV529 nucleic acid produced during replication. The relative infectious titre for each sample was determined using the parallel-line analysis as described in the European Pharmacopoeia 8.0
. The analysis by extrapolation is not an appropriate approach as several parameters including the similar conditions between the in-house reference control and test samples are not considered during analysis. In this study, the correlation between test samples and the in-house reference control was assessed using PLA software version 2.0. Before PLA analysis, all C
values for the in-house reference control and test samples were subjected to standard outlier analysis, with the limit that no more than one data point (one replicate out of the four replicates) per HSV529 dilution could be removed. Afterwards, each assay was analyzed by PLA software. The assay was considered valid if the regression, linearity, and parallelism were significant.
To investigate if RT-qPCR infectivity assay is a suitable method to evaluate the stability of HSV529 test samples, a concordance study was conducted between the RT-qPCR infectivity assay and a conventional infectivity plaque assay using identical test samples. While the results illustrated a suitable correlation (R2 ~0.91) between the qRT-PCR infectivity assay and the plaque assay, higher cost and complexity of RT-qPCR infectivity assay were two drawbacks of this method compare to a traditional method.
To evaluate the closeness of the analytically determined HSV529 infectious titre values, the accuracy of the method was evaluated in six independent assays by two analysts on different days. The accuracy was determined as the percentage of the infectious titre values obtained by RT-qPCR versus infectious titre values by a plaque assay. The accuracy was evaluated in the range of 92.91% to 120.57%, indicating a suitable accuracy for the assay. The intermediate precision of the assay was also evaluated to measure the variation of the obtained data. To evaluate this parameter, the assay was performed six times by two different operators over a time period of 2 months. The mean value of this run control was 16.53 log pfu/ml with a standard deviation of 0.091, resulting in a coefficient of variation of 9.19.