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

PRNT assay was performed, as previously described [31] , to determine the neutralizing capacity of elicited antibodies in sera from immunized BALB/C mice against MERS-CoV. Briefly, collected sera were firstly heated at 56 • C/30 min in water bath for inactivation. Sera were then two-fold serially diluted in 40 µL of DMEM/2% FBS (diluted from 1:20 to 1:160). To each sera dilution, an equal volume/amount of the plaque forming unit in 40 µL DMEM/2% FBS was supplied. After 1 h co-incubation of the serum/virus mixtures, 50 µL of each mixture were dispensed individually into 12-well tissue culture plates containing confluent monolayers of Vero-E6 cell. The cell monolayers were incubated together with the serum/virus mixtures at 37 • C for 1 h to allow virus adsorption. The infected Vero monolayers were washed with 1× PBS and supplied with agarose overlay containing 1× MEM media, 1% agarose, 1% Penicillin/Streptomycin (Pen/Strep), and then left to solidify. The plates were incubated until the formation of visible viral plaques (72 h). Cell monolayers were fixed with 3.4% formaldehyde solution for 1 h and stained with 1% crystal violet solution for 30 min at RT. Eventually, the plates were washed with water to visualize the plaques and the percent (%) of inhibition is calculated as following: % of plaque reduction = (virus control plaques count − sample plaques count)/(virus control plaques count) × 100.