The anti-M2e immune response was monitored by determining the titer of the M2e-specific IgY at three different time points (2 weeks after each inoculation). Chickens after each inoculation developed high levels of antibody against the injected construct and anamnestic response clearly was seen when the plates were coated with Mono-M2e and Tetra-M2e nanoparticles and M2e-GCN4 (tetrameric M2e), respectively (Table 1, Figures 7 and 8) . For further investigation of the antibodies, the level of M2e specific antibody was measured using plates coated with tetrameric M2e-GNC4 peptide to evaluate the specific antibody against tetrameric M2e rather than the whole particle. The result of this study indicated that in chickens, after the second booster, the antibody levels are not at the same level as our previous results in mice with the same backbone but a different (malaria) epitope had been shown [23] . The dose level was also higher than what was shown to be required in mice. This could be because of the lower haplotype-specific immunogenicity of the particles in chickens, the route of administration in mice (intraperitoneal and intranasally), the body weight of the mice compared with chickens, and different immune system repertoires of mammalian and avian species. In future studies, changing the administration route can be another approach to reducing the dose of vaccine construct. We also coated the plate with inactivated purified virus to observe seroconversion of the chickens after challenge with the virus at 2 weeks after the last boost. Results indicated that whole virus response was higher as expected with hyperimmune serum (Figure 9 (a)), however, ELISA response from chicken vaccinated with tetra-M2e and with whole virus reacted similarly on GCN-M2e coated plate (Figure 9(b) ). The protective efficacy of the anti-M2e antibody responses induced by different constructs was assessed by evaluation of viral shedding post challenge. To determine the peak of shedding, viral copy number was Figure 10 . We determined viral loads in tracheal and cloacal swabs samples on day 8 following challenge with 10 7.2 EID 50 LPAI subtype H5N2. Reduction of cloacal and oropharyngeal shedding in vaccinated birds was significant in chickens vaccinated with Tetra-M2e with Freund's adjuvant. Virus shedding was evaluated at day 4 and day 6 after challenge; the swabs were tested for virus load ( Figure 11 ). It is seen that virus shedding reduction starts at day 4 post infection with a significant decrease at day 8 post infection.