Prior to vaccination and challenge study, a pilot study was performed to evaluate pathogenicity Influenza Research and Treatment 5 Upon determination of the peak of virus shedding and appropriate infectious dose in the pilot study, the vaccination and challenge trial was initiated. Briefly, 42 SPF chickens were divided into six groups of seven and received their first inoculation at 2 weeks of age followed by two boosters, two weeks apart, at 10 weeks and 12 weeks after hatch as described in Table 2 . Preceding injection, nanoparticles were concentrated using Amicon Centrifugal Filter units with a 100 kDa MWCO (Millipore, Billerica, MA). Concentration was determined by absorbance at 280 nm and nanoparticle quality was assured by DLS. The nanoparticle vaccine constructs were emulsified with either Freund's complete adjuvant (prime) or Freund's incomplete adjuvant (boosters) and injected into the pectoral muscle of each chicken. Two weeks after the second booster, the birds, except for those in the negative control group, were challenged with 10 7.2 EID 50 LPAI subtype H5N2. Briefly, each bird received 1 mL allantoic fluid containing 10 7.2 EID 50 LPAI subtype H5N2 divided among the eyes, nasal cavity, and oropharynx, while temporarily blocking the fresh air delivery to the isolator. Fresh air was resumed after 5-10 min the following challenge of the last bird in the isolator. Although the clinical signs associated with LPAI viruses are rare, they were observed for possible clinical symptoms daily; and the presence of the symptoms and their severity was recorded.