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.
Figure 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),
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.