Male Fitch ferrets, 6-12 months of age (Triple F Farms, Sayre, PA), that were serologically negative by hemagglutinin inhibition (HAI) assay for currently circulating influenza viruses, were used in this study. All HA, NP and M2 genes are from A/Thailand/1(KAN-1)/2004. The numbers of animals used in our studies were as follows: (a) 4 animals for each group of NP and M2 alone, (b) 5 animals for the NP+M2 and the negative control group, (c) in another experiment, 4 animals for all three groups: HA alone, HA+NP+M2 and the vector control. The ferrets were immunized three times with a total of 250 mg plasmid DNA in 500 ml of PBS (pH 7.4) intramuscularly in the quadriceps muscle at 0, 3, and 6 weeks. For the single component plasmid group, each animal received 83 mg DNA for each plasmid with 167 mg of control vector as filler DNA (total 250 mg). For the 2 plasmid combination group (e.g. NP+M2), 83 mg of each NP and M2 plasmid with 83 mg control vector was used (total 250 mg). For the 3 plasmid combination group (HA+NP+M2), 83 mg of each three plasmid DNA was used as total DNA remained the same (total 250 mg). At week 9, the ferrets were immunized intramuscularly with 10 10 particles of recombinant adenoviruses expressing HA, NP, M2, or in different combinations similar to their DNA immunization combinations. Serum was collected 10 days after the last vaccination. The DNA and adenovirus immunizations were conducted at BioQual, Inc. (Rockville, MD). Nine to ten weeks after the adenovirus boost, the immunized ferrets were challenged with A/Vietnam/1203/2004 virus, which has the identical NP and M2 amino acid sequence as that of the immunized strain A/Thailand/1(KAN-1)/2004, at the Centers for Disease Control and Prevention (Atlanta, GA) as previously described [19] . Briefly, 2 days prior to infection, baseline serum, body temperature, and weight measurements of the ferrets were obtained. After the ferrets were anesthetized by an intramuscular injection of ketamine hydrochloride (24 mg/kg), xylazine (2 mg/kg) and atropine (0.05 mg/kg) cocktail, they were inoculated intranasally with 10 7 EID 50 of virus in 1 ml of PBS. The ferrets were monitored for changes in body temperature and weight and the presence of the following clinical symptoms: sneezing, lethargy, anorexia, nasal or ocular discharge, dyspnea, diarrhea, and neurological dysfunction. Body temperatures were measured using an implantable subcutaneous temperature transponder (BioMedic Data Systems, Inc., Seaford, DE). Viral titers were measured in nasal washes collected on days 3, 5, and 7 post-infection from anesthetized ferrets as previously described [6] . The nasal washes were immediately frozen on dry ice and stored at 270uC until they were processed. Viral titers in the nasal washes were determined in eggs as described above. Any ferret that lost more than 25% of its body weight or exhibited neurological dysfunction was euthanized and submitted to postmortem examination. Body weight, clinical symptoms, signs of morbidity, and survival were monitored for 7 or up to 13 days. The statistical significance of differences in survival between groups was determined using a log-rank test.