Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines

Abstract

Source: Pérez-Girón, J. V. et al, Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity. J. Vis. Exp. (2015)

The video showcases a method to create cold-adapted recombinant influenza viruses. It involves transfecting human cells with bi-directional plasmids, followed by incubation at lower temperature incubation to yield viruses suitable for vaccine production, characterized by reduced replication in warmer conditions.

Protocol

1. Generation of Recombinant Live Attenuated Influenza Vaccines by Reverse Genetics

NOTE: The detailed protocol for the generation of recombinant influenza viruses by reverse genetics has been described by previous studies and is out of the scope of this report. Briefly, rescue of cold-adapted influenza vaccines (CAV) is done in a biosafety class II cabinet under biosafety level 2 (BSL2) containment, and involves steps detailed below.

1. Generate a reassortant cold-adapted influenza vaccine using as background the cold-adapted strain A/Ann Arbor/6/60 (H2N2), as well as the hemagglutinin (HA) and a ....

Results

Figure 1. Engineering of traceable LAIVs. (A) Influenza segments cloned in ambisense plasmids were transfected into 293T cells using Lipofectamine 2000. 24 hr after transfection, supernatants obtained from 293T cells were used to coat influe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM 1X)	Gibco RL-Life Technologies	41965-039
Opti MEM	Gibco RL-Life Technologies	31985-047
Lipofectamine 2000	Invitrogen-Life Technologies	11668-027
Penicillin-Streptomycin (10.000 U/ml)	PAA	p11-010
Bovine Serum Albumin	Sigma-Aldrich	A2153
Dymethil-Sulfoxide (DMSO)	Sigma-Aldrich	D2650
Trypsin-TPCK	Sigma-Aldrich	T1426