High throughput AS LNA qPCR method for the detection of a specific mutation in poliovirus vaccine strains.

Sabin Inactivated Poliovirus Vaccine (sIPV) has become one of the preferred vaccination options for the last step in the Poliovirus eradication program. Sequencing of poliovirus samples is needed during the manufacturing of poliovirus vaccines to assure the safety and immunogenicity of these vaccines. Next-generation sequencing analysis is the current costly and time-consuming gold standard for monitoring the manufacturing processes. We developed a low-cost and quick, highly sensitive, and allele-specific locked nucleic acid-probe-based reverse transcription quantitative PCR alternative that can accurately detect mutations in poliovirus vaccine samples during process development, scaling up, and release. Using the frequently in vitro occurring and viral replication-impacting VP1-E295K mutation as a showcase, we show that this technology can accurately detect E295K mutations in poliovirus 2 samples to similar levels as NGS. The qPCR technology was developed employing a synthetic dsDNA fragment-based standard curve containing mixes of E295K-WT (wildtype) and Mut (mutant) synthetic dsDNA fragments ranging from 1 × 107 copies/µL to 1 × 102 copies/µL to achieve a linear correlation with R2 > 0.999, and PCR efficiencies of 95-105 %. Individual standard concentration levels achieved accuracies of ≥92 % (average 96 %) and precisions of ≤17 % (average 3.3 %) RSD. Specificity of locked nucleic acid (LNA)-probes was confirmed in the presence and absence of co-mutations in the probe-binding region. Application of the developed assay to Sabin Poliovirus type 2 production run samples, illustrated a linear relationship with an R2 of 0.994, and an average accuracy of 97.2 % of the variant (allele)-specific AS LNA qPCR result, compared to NGS. The assay showed good sensitivity for poliovirus samples, containing E295K mutation levels between 0 % and 95 % (quantification range). In conclusion, the developed AS LNA qPCR presents a valuable low-cost, and fast tool, suitable for the process development and quality control of polio vaccines.

Identification of probable early-onset biomarkers for tuberculosis disease progression.

Determining what constitutes protective immunity to TB is critical for the development of improved diagnostics and vaccines. The comparison of the immune system between contacts of TB patients, who later develop TB disease (progressors), versus contacts who remain healthy (non-progressors), allows for identification of predictive markers of TB disease. This study provides the first comprehensive analysis of the immune system of progressors and non-progressors using a well-characterised TB case-contact (TBCC) platform in The Gambia, West Africa. 22 progressors and 31 non-progressors were analysed at recruitment, 3 months and 18 months (time to progression: median[IQR] of 507[187-714] days). Immunophenotyping of PBMC, plasma cytokine levels and RT-MLPA analysis of whole blood-derived RNA was performed to capture key immune system parameters. At recruitment, progressors had lower PBMC proportions of CD4+ T cells, NKT cells and B cells relative to non-progressors. Analysis of the plasma showed higher levels of IL-18 in progressors compared to non-progressors and analysis of the RNA showed significantly lower gene expression of Bcl2 but higher CCR7 in progressors compared to non-progressors. This study shows several markers that may predict the onset of active TB at a very early stage after infection. Once these markers have been validated in larger studies, they provide avenues to prospectively identify people at risk of developing TB, a key issue in the testing of new TB vaccines.