Expression of H5 hemagglutinin vaccine antigen in common duckweed ( Lemna minor ) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens

Abstract A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (r Lemna -HA). In Experiment 1, efficacy of r Lemna -HA was tested on birds immunized with 0.2 μg or 2.3 μg HA and challenged with 106 mean chicken embryo infectious doses (EID50 ) of homologous virus strain. Both dosages of r Lemna -HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of r Lemna -HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of r Lemna -HA was tested on birds immunized with 0.9 μg or 2.2 μg HA and challenged with 106 EID50 of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9 μg HA and 2.2 μg HA, respectively. For each challenge virus, viral shedding titers from 2.2 μg HA vaccinated birds were significantly lower than those from 0.9 μg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (r Lemna -HA 0.9 μg HA) and 40% (r Lemna -HA 2.2 μg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna -expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a good alternative for producing high quality antigen for an injectable vaccine against H5N1 HPAI viruses.