(A) Plaque phenotypes on MDCK cells of PR8 wild type virus and synthetic PR8 derivatives, carrying one (NP^Min, HA^Min, PB1^Min), two (NP/HA^Min; HA/PB1^Min) or three (PR8^3F) deoptimized gene segments. (B) Growth kinetics of PR8 wild type virus and three synthetic PR8 derivatives in MDCK cells after infection with 0.001 MOI of the indicated viruses. All combinations grew within log 8-9 although only PR8-HA^Min, PR8-NP/HA^Min and PR8^3F are shown as compared to wild type. (C) Analysis of Influenza virus protein expression in infected cells. MDCK cells were infected with PR8 wild type virus or synthetic PR8 derivatives, carrying one deoptimized gene segment each (NP^Min, HA^Min, PB1^Min), as indicated. Western blot (WB) analysis of proteins extracted from whole cell lysates was carried out with PB1, NP, HA, or actin antibodies. Actin was used to indicate equal protein loading.

(A) Body weight curve following intranasal infection with 10⁴ PFU of PR8 wild type (triangles), 10⁴ PFU of deoptimized PR8^3F (diamonds), or mock-infected (saline; squares). The average of 5 mice per time point and standard deviations are indicated. Wild type-infected mice did not survive beyond day 5 (indicated by a cross). (B) Virus titer in whole lung homogenate after infection with either 10³ PFU PR8 wild type (squares), or deoptimized PR8^3F (circles). Average of three mice per time point. * On day 9 post infection, PR8^3F was no longer detectable (below 40 PFU/lung).

(A, B) Vaccine Margin of Safety for PR8 wild type and deoptimized PR8^3F viruses. The left ordinate indicates the percentage of animals surviving the primary inoculation (black squares) with (A) PR8^3F or (B) wt PR8, at doses ranging between 10⁰ to 10⁶ PFU. After 28 days, the surviving, vaccinated animals were challenged with a single 1000 × lethal dose 50 of PR8 wild type virus. Disease and survival were monitored (right ordinate; open circles) for (A) PR8^3F- and (B) PR8-vaccinated mice. (C) Virus load in mouse lungs following wild type challenge of PR8^3F-vaccinated animals. 28 days following a single intranasal vaccination with 10⁴ PFU PR8^3F mice were challenged with 1000 × LD₅₀ of PR8 wt virus. Three days thereafter the level of challenge virus in lung homogenates was determined. (D) ELSIA determination of influenza-specific serum antibodies. 28 days after a primary infection, serum was collected, and anti-influenza IgG serum antibody titers were determined from animals that had received a primary inoculation of 0.01 × LD₅₀ (black diamonds) or 0.001 × LD₅₀ of PR8^3F (black circles), 0.01 × LD₅₀ of PR8 (white squares), or saline (black triangles). ELISA antibody titer against PR8 virus antigen is expressed as the lowest reciprocal serum dilution that resulted in a positive ELISA signal (5 standard deviation above background).