Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine.

Victoria Matyushenko,Irina Isakova-Sivak,Tatiana Tretiak,Irina Dubrovina,Larisa Rudenko,Tatiana Smolonogina

doi:10.1371/journal.pone.0180497

Victoria Matyushenko, Irina Isakova-Sivak + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0180497

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Journal: PloS one	Publication Date: Jul 7, 2017
Citations: 9	License type: CC BY 4.0

Affiliation: Institute of Experimental Medicine

Abstract

Live attenuated influenza vaccines (LAIVs) are considered as safe and effective tool to control influenza in different age groups, especially in young children. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with special attention to a potential virus transmission to the unvaccinated close contacts. Conducting LAIV clinical trials in some geographical regions with year-round circulation of influenza viruses warrants the development of robust and reliable tools for differentiating vaccine viruses from wild-type influenza viruses in nasal pharyngeal wash (NPW) specimens of vaccinated subjects. Here we report the development of genotyping assay for the detection of wild-type and vaccine-type influenza virus genes in NPW specimens of young children immunized with Russian-backbone seasonal trivalent LAIV using Sanger sequencing from newly designed universal primers. The new primer set allowed amplification and sequencing of short fragments of viral genes in NPW specimens and appeared to be more sensitive than conventional real-time RT-PCR protocols routinely used for the detection and typing/subtyping of influenza virus in humans. Furthermore, the new assay is capable of defining the origin of wild-type influenza virus through BLAST search with the generated sequences of viral genes fragments.

Highlights

Influenza is a vaccine preventable disease, severe influenza is still a major cause of early childhood deaths in low-income countries [1], mostly due to the unavailability of cheap, safe and effective influenza vaccines in these countries
Various assays have been previously developed to distinguish wild-type and vaccine-type viruses during the process of Live attenuated influenza vaccines (LAIVs) reassortant strain preparation, known as genome composition analysis [16,17,18,19,20]
Of them only “partial sequencing” strategy could be employed to differentiate LAIV virus from unknown wild-type virus in a field clinical trials because this assay is based on the amplification of viral gene from primers annealing to very conservative gene regions [19]

Summary

Introduction

Influenza is a vaccine preventable disease, severe influenza is still a major cause of early childhood deaths in low-income countries [1], mostly due to the unavailability of cheap, safe and effective influenza vaccines in these countries. A study evaluating safety and immunogenicity of a trivalent seasonal Russian-backbone live attenuated influenza vaccine (LAIV) in children 2 to 5 years of age was conducted in Bangladesh. The vaccine was proved to be safe for this age group without evidence of increased frequencies of adverse events in a vaccine cohort compared to a placebo group [2]. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with.

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