Fig. 3. Immune responses and protection.

(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).