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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 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.
- Plate 106 293T cells per well in 6-well plates using DMEM 10% fetal bovine serum (FBS) supplemented with 1% penicillin/streptomycin (P/E).
- 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).
- Incubate transfection mix for 30 min at RT.
- Add 100 µl of transfection mix/well and incubate for 16 hr at 37 °C and 5% CO2.
- Change cell media by DMEM 0.3% BSA 1% P/E at 33 °C and 5% CO2 for 5 hr.
- 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.
- Collect supernatants of MDCK-293T cell co-cultures and clear by centrifugation at 260 x g for 5 min.
