Smith, TanjaO’Kennedy, Martha M.Wandrag, D.B.R. (Daniel)Adeyemi, ModupeoreAbolnik, Celia2020-03-132020-03-132020-02Smith, T., O'Kennedy, M.M., Wandrag, D.B.R. et al. 2020, 'Efficacy of a plant-produced virus-like particle vaccine in chickens challenged with Influenza A H6N2 virus', Plant Biotechnology Journal, vol. 18, pp. 502-512.1467-7644 (print)1467-7652 (online)10.1111/pbi.13219http://hdl.handle.net/2263/73743Figure S1 Multiple sequence alignment of the hemagglutinin (HA) proteins of the strains used in this study.Figure S2 LC-MS/MS-based peptide sequence analysis for SDSPAGE bands of approximately 62 kDa (A) and 14 kDa (B), respectively.Table S1 Pairwise amino acid distances of the hemagglutinin proteins of H6N2 strains used in the study.Table S2 qRT-PCR results for oropharyngeal swabs as log10 vRNA viral titres/mL, with EID50/mL titres in parenthesis.Table S3 qRT-PCR results for cloacal swabs as log10 vRNA viral titres/mL, with EID50/mL titres in parenthesis.The efficacy, safety, speed, scalability and cost-effectiveness of producing hemagglutinin-based virus-like particle (VLP) vaccines in plants are well-established for human influenza, but untested for the massive poultry influenza vaccine market that remains dominated by traditional egggrown oil-emulsion whole inactivated virus vaccines. For optimal efficacy, a vaccine should be closely antigenically matched to the field strain, requiring that influenza A vaccines be updated regularly. In this study, an H6 subtype VLP transiently expressed in Nicotiana benthamiana was formulated into a vaccine and evaluated for efficacy in chickens against challenge with a heterologous H6N2 virus. A single dose of the plant-produced H6 VLP vaccine elicited an immune response comparable to two doses of a commercial inactivated H6N2 vaccine, with mean hemagglutination inhibition titres of 9.3 log2 and 8.8 log2, respectively. Compared to the non-vaccinated control, the H6 VLP vaccine significantly reduced the proportion of shedders and the magnitude of viral shedding by >100-fold in the oropharynx and >6-fold in the cloaca, and shortened oropharyngeal viral shedding by at least a week. Despite its potency, the cost of the antigenic mismatch between the inactivated H6N2 vaccine and challenge strain was evident not only in this vaccine’s failure to reduce viral shedding compared to the non-vaccinated group, but its apparent exacerbation of oropharyngeal viral shedding until 21 days post-challenge. We estimate that a kilogram of plant leaf material can produce H6 VLP vaccines sufficient for between 5000 and 30 000 chickens, depending on the effective dose and whether one or two immunizations are administered.en© 2019 The Authors. This is an open access article distributed under the terms of the Creative Commons Attribution License.Avian influenzaNicotiana benthamianaVLP vaccineH6N2ChickensViral sheddingVirus-like particle (VLP)Article