Newcastle disease (ND) is a highly contagious avian disease, causing considerable economic losses to the poultry industry. To obtain a safe, inexpensive, and effective ND vaccine to meet the international trade requirements of differentiating infected from vaccinated animals (DIVA), here we report the production of Oryza sativa recombinant fusion (F) protein in stably transformed transgenic rice seeds via agroinfiltration. The F protein expression level was enhanced 3.6-fold with a genetic background in low glutelin. Inoculation of plant-produced F antigen into Specific Pathogen Free (SPF) chickens markedly elicited neutralizing antibody responses against homologous and heterologous ND virus strains. Two doses of 4.5 μg fully protected chickens from a lethal ND challenge without any clinical symptoms. The mean weight gain of F protein-immunized chickens within 15 days after challenge was significantly higher than that of traditional whole virus vaccine-immunized chickens, thereby obtaining higher economic benefits. Moreover, the sera from the chickens vaccinated with the plant-produced F vaccine did not show reactivity in an immunochromatographic strip targeting the haemagglutinin-neuraminidase protein (HN) protein, and DIVA could be achieved within 10 minutes. Our results demonstrate that the plant-derived F vaccine along with immunochromatographic strips could be useful in the implementation of an NDV eradication program.

Summary: Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone‐dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 μg provided effective protection in pigs without interference from pre‐existing antibodies. Crystal structure and small‐angle X‐ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

The H9N2 avian influenza virus (AIV) remains a serious threat to the global poultry industry and public health. The hemagglutinin (HA) protein is an essential protective antigen of AIVs and a major target of neutralizing antibodies and vaccines. Therefore, in this study, we used rice-derived HA protein as an immunogen to generate monoclonal antibodies (mAbs) and screened them using an immunoperoxidase monolayer assay and indirect enzyme-linked immunosorbent assay. Eight mAbs reacted well with the recombinant H9N2 AIV and HA protein, four of which exhibited potent inhibitory activity against hemagglutination, while three showed remarkable neutralization capacities. Western blotting confirmed that two mAbs bound to the HA protein. Linear epitopes were identified using the mAbs; a novel linear epitope, ⁴⁸⁰HKCDDQCM⁴⁸⁷, was identified. Structural analysis revealed that the novel linear epitope is located at the C-terminus of HA2 near the disulfide bond-linked HA1 and HA2. Alignment of the amino acid sequences showed that the epitope was highly conserved among multiple H9N2 AIV strains. The results of this study provide novel insights for refining vaccine and diagnostic strategies and expand our understanding of the immune response against AIV.

The E2 protein of classical swine fever virus (CSFV) was expressed in transgenic rice endosperms as a diagnostic antigen for use with a rapid colloidal gold assay for the detection of antibodies (Abs) against CSFV. This improved test was used to monitor Abs against the E2 protein in both pigs and rabbits immunized with a live attenuated vaccine or E2 subunit vaccine.

The authors would like to make the following correction to this published paper