Phiv luciferase

The serum neutralizing antibody titers in immunized mice were measured as previously described. Briefly, pseudotyped SARS-CoV-2 viruses were obtained by co-transfection of packaging plasmid psPAX2 (Addgene), luciferase-expressing lentivirus plasmid pHIV-Luciferase (Addgene) and plasmid expressing SARS-CoV-2 spike protein (isolate Wuhan-Hu-1, GenBank: QHD43416.1) into HEK293T cells. Then serially diluted serum of BALB/c mice were mixed with pseudotyped SARS-CoV-2 viruses and incubated at 37°C for 1 hour. Serum/virus mixtures were added into wells which were seeded with hACE2-HEK293T cells and went on culturing for 48 h. After 48 h, cells were lysed with lysis buffer (Promega) and were measured for relative luminescence units in luminometer (Promega).

The IAV subtypes used in this study, including the pH1N1 (A/England/2009) and H3N2 (A/HK/1999), were provided by Wendy Barclay (Imperial College, London) and Leo Poon (University of Hong Kong), respectively. The plasmids used for the production of H1N1 and H3N2 pseudo-typed viral particles were obtained from Addgene; pHIV-Luciferase (Addgene plasmid #21375); psPAX2 (Addgene plasmid # 12260); and Vesicular Stomatitis Virus (VSV-G) (Addgene plasmid #8454). pcDNA3.1-swineH1-flag (H1 from swine H1N1 A/California/04/09) (Invitrogen), pcDNA3.1-swine N1-flag (N1 from swine H1N1 A/California/04/09), and pCDNA H3 (from A/Denmark/70/03 (H3N2) (Invitrogen) were obtained commercially. pI.18-N2 [N2 from A/Texas/50/2012/(H3N2)] plasmid was a gift from Nigel Temperton (University of Kent). Anti-influenza antibodies used were obtained from BEI Resources, NIAID, NIH, USA, and used as previously described (38 (link)); these include polyclonal anti-influenza Virus H3 HA, A/Hong Kong/1/1968 and monoclonal anti-influenza virus H1 HA, A/California /04/2009.

The spike-expressing plasmids including pcDNA3.1-SARS-CoV-2-S-WT (D614; Wuhan-Hu-1), pcDNA3.1-SARS-CoV-2-S-Alpha (B.1.1.7), pcDNA3.1-SARS-CoV-2-S-Beta (B.1.351), pcDNA3.1-SARS-CoV-2-S-Gamma (P.1), pcDNA3.1-SARS-CoV-2-S-Epsilon (B.1.429), pcDNA3.1-SARS-CoV-2-S-lota (B.1.526), pcDNA3.1-SARS-CoV-2-S-Kappa (B.1.617.1), pcDNA3.1-SARS-CoV-2-S-Delta (B.1.617.2), pcDNA3.1-SARS-CoV-2-S-Lambda (C.37), pcDNA3.1-SARS-CoV-2-S-Omicron (BA.1, BA.2, BA.2.12.1, BA.4, XBB, and BQ.1.1), pcDNA3.1-SARS-CoV-S, and pcDNA3.1-MERS-CoV-S were synthesized or preserved in our laboratory.
To produce pseudovirions, HEK293T cells were co-transfected with a plasmid expressing S protein as described above, a backbone plasmid pHIV-Luciferase (Addgene plasmid #21375) that encoded luciferase reporter under the control of EF-1α promoter, and a packaging construct psPAX2 (Addgene plasmid #12260). The cell culture supernatants containing the released virions were collected at 72 h post-transfection, passed through a 0.45 μΜ filter, and frozen at −80 °C.