Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHA H5N6 + H9N2 BLP or a combination of tHA H5N6 BLP and tHA H9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

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Summary The ongoing coronavirus disease 2019 (COVID‐19) pandemic has spurred rapid development of vaccines as part of the public health response. However, the general strategy used to construct recombinant trimeric severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike (S) proteins in mammalian cells is not completely adaptive to molecular farming. Therefore, we generated several constructs of recombinant S proteins for high expression in Nicotiana benthamiana . Intramuscular injection of N. benthamiana ‐expressed S ct vaccine (NS ct Vac) into Balb/c mice elicited both humoral and cellular immune responses, and booster doses increased neutralizing antibody titres. In human angiotensin‐converting enzyme knock‐in mice, two doses of NS ct Vac induced anti‐S and neutralizing antibodies, which cross‐neutralized Alpha, Beta, Delta and Omicron variants. Survival rates after lethal challenge with SARS‐CoV‐2 were up to 80%, without significant body weight loss, and viral titres in lung tissue fell rapidly, with no infectious virus detectable at 7‐day post‐infection. Thus, plant‐derived NS ct Vac could be a candidate COVID‐19 vaccine.

Poly(A)-binding protein (PABP) is a translation initiation factor that interacts with the poly(A) tail of mRNAs. PABP bound to poly(A) stimulates translation by interacting with the eukaryotic initiation factor 4G (eIF4G), which brings the 3′ end of an mRNA close to its 5′ m7G cap structure through consecutive interactions of the 3′-poly(A)–PABP-eIF4G-eIF4E-5′ m7G cap. PABP is a highly abundant translation factor present in considerably larger quantities than mRNA and eIF4G in cells. However, it has not been elucidated how eIF4G, present in limited cellular concentrations, is not sequestered by mRNA-free PABP, present at high cellular concentrations, but associates with PABP complexed with the poly(A) tail of an mRNA. Here, we report that RNA-free PABPs dimerize with a head-to-head type configuration of PABP, which interferes in the interaction between PABP and eIF4G. We identified the domains of PABP responsible for PABP–PABP interaction. Poly(A) RNA was shown to convert the PABP–PABP complex into a poly(A)–PABP complex, with a head-to-tail-type configuration of PABP that facilitates the interaction between PABP and eIF4G. Lastly, we showed that the transition from the PABP dimer to the poly(A)–PABP complex is necessary for the translational activation function.

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