Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production

Abstract

Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012–2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8 EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1 EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate.

Human bone marrow‐derived MSCs spontaneously express specific Schwann cell markers

“…The linear graph was generated, and the PCR efficiency was determined from the curve. PCR efficiency between 90% and 110% was obtained for all the primers, which corresponds to a slope of between −3.58 and −3.10 (Shcherbik et al, ).…”

Use of a pandemic H1N1 strain with updated haemagglutinin and neuraminidase results in increased nasopharyngeal shedding and improved immunogenicity to Russian-backbone live attenuated influenza vaccine among children aged 2 – 4 years old: an open-label, prospective, observational, phase 4 study in The Gambia

“…RT-PCR assays with primers and probes mapping to internal genes were used to distinguish LAIV from seasonal influenza viruses (appendix, table S4). 13 Despite optimisation of assay conditions, maximum LAIV dilutions detected by LAIV-specific RT-PCR were at least one log10 lower than those detected by HAspecific RT-PCR (Table S5). Only samples with ct values of £30 in seasonal influenza assays were therefore tested with LAIV-specific assays, with 100% confirmed as LAIV strains.…”

Разработка Холодоадаптированного Реассортантного Вируса Гриппа А/H6n1 На Основе Донора Аттенуации a/Ленинград/134/17/57 (H2N2) И Его Генотипирование Методом Анализа Кривых Плавления Высокого Разрешения (HRM-анализ)

“…Для быстрого анализа состава генома потенциальных вакцинных кандидатов применяют также ПЦР в реальном време-ни с использованием гидролизируемых олигонукле-отидных зондов [12] и пиросеквенирования [13]. Для упрощения процедуры скрининга вакцинных кан-дидатов мы предлагаем использовать ПЦР в режиме реального времени с анализом кривых плавления вы-сокого разрешения (HRM-анализ).…”