Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines

Published: May 31, 2024

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. Generate…

Representative 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 influenza-permissive Madin-Darby canine kidney (MDCK) cells in the presence of 1% of TPCK trypsin. All the protocol was performed at…

Offenlegungen

The authors have nothing to disclose.

Materials

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

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Diesen Artikel zitieren
Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines. J. Vis. Exp. (Pending Publication), e22254, doi: (2024).

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