Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines

プロトコル

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 modified neuraminidase (NA) of the A/PR/8/34 (H1N1) strain (Figure 1A). The modification in the NA gene consists of a PCR-based replacement of a conserved sequence in the protein stalk (residues 65 to 72) by a short cDNA sequence encoding the chicken ovalbumin (OVA)-derived peptide SIINFEKL (Figure 1B). The SIINFEKL peptide is a highly immunodominant peptide in the context of the H-2b MHC class I-restricted response of C57BL/6 mice.
2. Plate 10⁶ 293T cells per well in 6-well plates using DMEM 10% fetal bovine serum (FBS) supplemented with 1% penicillin/streptomycin (P/E).
3. Prepare plasmid transfection mixture: Add 1 µg of each one of the plasmids encoding cDNAs for PB2, PB1, PA, NP, M, NS from the influenza A/Ann Arbor/6/60 strain and 1 µg of the plasmids encoding for the NA-SIINFEKL and HA from influenza A/PR/8/34. Add pre-warmed Opti- minimal essential medium (Opti-MEM) (15 µl for a final volume of 100 µl/well).
4. Incubate transfection mix for 30 min at RT.
5. Add 100 µl of transfection mix/well and incubate for 16 hr at 37 °C and 5% CO₂.
6. Change cell media by DMEM 0.3% BSA 1% P/E at 33 °C and 5% CO₂ for 5 hr.
7. Add 10⁶ influenza permissive Madin-Darby Canine Kidney (MDCK) cells in dimethyl sulfoxide (DMSO) containing 1 µg/ml of trypsin from bovine pancreas treated with L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK-trypsin). Maintain the 293T-MDCK co-cultures for 2 - 3 days at 33 °C and 5% CO₂.
8. Collect supernatants of MDCK-293T cell co-cultures and clear by centrifugation at 260 x g for 5 min.

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結果

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 33 °C. Viral supernatants from MDCK cells were then inoculated into 9-day-old embryonated chicken eggs for three days at 33 °C. Viral rescue was confirmed by PCR-based viral genome amplification and sequencing. (B) To insert the OVA-derived SIINFEKL peptide into the influenza neuraminidase (NA), a conserved region (residues 65 to 72) of the NA gene of A/Puerto Rico/8/34 (H1N1) virus was substituted for a short cDNA encoding the SIINFEKL peptide via fusion PCR.

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資料

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