A Recombinant Influenza A/H1N1 Carrying A Short Immunogenic Peptide of MERS-CoV as Bivalent Vaccine in BALB/c Mice

Shehata, Mahmoud M.; Kandeil, Ahmed; Mostafa, Ahmed; Mahmoud, Sara H.; Gomaa, Mokhtar R.; El-Shesheny, Rabeh; Webby, Richard; Kayali, Ghazi; A. Ali, Mohamed

Using the eight-plasmid reverse genetics system, recombinant rgH1N1pdm09 (WT 5 + 3), and rgH1N1-MERS-CoV (chimeric bivalent 5 + 3) viruses were generated using (pHW-PB2PR8, pHW-PB1H1N1pdm09, pHW-PAPR8, pHW-HAH1N1pdm09, pHW-NPPR8, pHW-NAH1N1pdm09, pHW-MPR8, and pHW-NSPR8) and (pHW-PB2PR8, pHW-PB1H1N1pdm09, pHW-PAPR8, pHW-HAH1N1pdm09, pHW-NPPR8, pHW-NAH1N1pdm09-MERS-CoV, pHW-MPR8, and pHW-NSPR8), respectively, as described previously [29] . Briefly, the day before transfection, 293T/MDCK-II cells were co-cultured in a ratio of 3/1 then incubated at 37 • C, 5% CO 2 . On the day of transfection, 8 µg of the eight desired DNA plasmids (1 µg per plasmid) was mixed with Trans-IT2020 (Mirus, Madison, WI, USA) (2 µL per 1 µg plasmid) and 184 µL of Opti-MEM reduced serum medium (Invitrogen, Carlsbad, CA, USA) and incubated for 45 min at room temperature. Afterwards, a volume of 800 µL of Opti-MEM medium was added to the mixture. The mixture was added drop wise to 293T/MDCK-II cells then incubated at 37 • C, 5% CO 2 for 6 h. The transfection mixture was replaced with 1 mL Opti-MEM containing 1% antibiotic-antimycotic mixture, and 0.4% bovine serum albumin, and the cells were incubated overnight at 37 • C,5 % CO 2 . Subsequently, an additional 1 mL Opti-MEM containing Pen/Strep, 0.2% bovine serum albumin (BSA), and 2 µg L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich, St. Louis, MI, USA) was added and the cells were further incubated for 48 h. The culture supernatants were harvested and clarified by low-speed centrifugation. Then, we injected the supernatant into the 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs and MDCK-II cells. Standard hemagglutination test (HA) was used to detect the recombinant viruses in the allantoic fluid of embryonated chicken eggs and MDCK-II cells harvest. Recombinant viruses were harvested and propagated in SPF embryonated chicken eggs for three passages. The NA genes of the generated rescued viruses were amplified and sequenced to test stability of the inserted short peptide.