All animal research was conducted under the guidance of the Centers for Disease Control and Prevention's Institutional Animal Care and Use Committee in an animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Female BALB/c mice, 6-8 weeks old (Jackson Labs, Bar Harbor, ME), were immunized as previously described [22] with HA from A/Thailand/1(KAN-1)/2004, NP from A/PR/8/34, and M2 from A/Thailand/1(KAN-1)/2004. Briefly, mice (10 animals for each group of HA alone, HA+NP, and HA+NP+M2; and 5 animals for the NP alone and vector control) were immunized three times with a total of 15 mg plasmid DNA in 100 ml of PBS (pH 7.4) intramuscularly at 0, 3, and 6 weeks. For the single HA or NP plasmid group, each group received 5 mg DNA for each plasmid with 10 mg of control vector as filler DNA (total 15 mg). For the 2 plasmid combination group (HA+NP), 5 mg of each plasmid plus 5 mg control vector was used. For the 3 plasmid combination group (HA+NP+M2), 5 mg of each plasmid DNA was used as total DNA remained the same (15 mg). Serum was collected 10 days after the last DNA vaccination.