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…
Disclosures
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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Cite This Article
Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines. J. Vis. Exp. (Pending Publication), e22254, doi: (2024).