Immunization with Plant-Derived Multimeric H5 Hemagglutinins Protect Chicken against Highly Pathogenic Avian Influenza Virus H5N1.

Since 2003, H5N1 highly pathogenic avian influenza viruses (HPAIV) have not only caused outbreaks in poultry but were also transmitted to humans with high mortality rates. Vaccination is an efficient and economical means of increasing immunity against infections to decrease the shedding of infectious agents in immunized animals and to reduce the probability of further infections. Subunit vaccines from plants are the focus of modern vaccine developments. In this study, plant-made hemagglutinin (H5) trimers were purified from transiently transformed N. benthamiana plants. All chickens immunized with purified H5 trimers were fully protected against the severe HPAIV H5N1 challenge. We further developed a proof-of-principle approach by using disulfide bonds, homoantiparallel peptides or homodimer proteins to combine H5 trimers leading to production of H5 oligomers. Mice vaccinated with crude leaf extracts containing H5 oligomers induced neutralizing antibodies better than those induced by crude leaf extracts containing trimers. As a major result, eleven out of twelve chickens (92%) immunized with adjuvanted H5 oligomer crude extracts were protected from lethal disease while nine out of twelve chickens (75%) vaccinated with adjuvanted H5 trimer crude extracts survived. The solid protective immune response achieved by immunization with crude extracts and the stability of the oligomers form the basis for the development of inexpensive protective veterinary vaccines.

In vitro-Formulated Oligomers of Strep-Tagged Avian Influenza Haemagglutinin Produced in Plants Cause Neutralizing Immune Responses.

The worldwide emergence of the novel influenza A H5N1 and H5N8 has notably and directly impacted the poultry industry, resulting in the need for effective and cheap vaccination strategies to protect poultry worldwide. Subunit vaccines from plants can be produced for a low cost, and plant production systems are easily scaled up at low infrastructure cost. However, subunit vaccines generally induce low immunogenicity against influenza. To address this issue, we present a new and innovative method to generate highly immunogenic H5 oligomers. The method is based on specific and high-affinity interaction between engineered streptavidin (Strep-Tactin® XT) and the Strep-tag II peptide. H5-Strep-tag II-tagged trimers were produced via transient agroinfection in tobacco leaves and purified, and oligomers were formulated in vitro by adding purified homotetrameric Strep-Tactin® XT. Immunogenicity was tested by performing mouse immunizations. Haemagglutinin oligomers produced in vitro by combining Strep-Tactin® XT and Strep-tag II-fused haemagglutinin trimers from plants raised potentially neutralizing antibodies in mice. Vaccines based on actual H5N1 haemagglutinin can be produced by combining strep-tagged haemagglutinin trimers from plants and Strep-Tactin® XT.

Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants.

Plant-based transient expression is an alternative platform to produce hemagglutinin-based subunit vaccines. This production system provides not only fast and effective response in the context of a pandemic but also enables the supply of big volume vaccines at low cost. Crude plant extracts containing influenza hemagglutinin are considered to use as vaccine sources because of avoidance of related purification steps resulting in low cost production allowing veterinary applications. Highly immunogenic influenza hemagglutinins are urgently required to meet these pre-conditions. Here, we present a new and innovative way to generate functional H5 oligomers from avian flu hemagglutinin in planta by the specific interaction of S·Tag and S·Protein. A S·Tag was fused to H5 trimers and this construct was transiently co-expressed in planta with S·Protein-TPs which was multimerized by disulfide bonds via cysteine residues in tailpiece sequences (TP) of IgM antibody. Multimerized S·Protein-TPs serve as bridges/molecular docks to combine S·Tag-fused hemagglutinin trimers to form very large hemagglutinin H5 oligomers. H5 oligomers in the plant crude extract were highly active in hemagglutination resulting in high titers. Immunization of mice with two doses of plant crude extracts containing H5 oligomers after storage for 1 week at 4 °C caused strong immune responses and induced neutralizing specific humoral immune responses in mice. These results allow for the development of cheap influenza vaccines for veterinary application in future.

The Encapsulation of Hemagglutinin in Protein Bodies Achieves a Stronger Immune Response in Mice than the Soluble Antigen.

Zein is a water-insoluble polymer from maize seeds that has been widely used to produce carrier particles for the delivery of therapeutic molecules. We encapsulated a recombinant model vaccine antigen in newly formed zein bodies in planta by generating a fusion construct comprising the ectodomain of hemagglutinin subtype 5 and the N-terminal part of γ-zein. The chimeric protein was transiently produced in tobacco leaves, and H5-containing protein bodies (PBs) were used to immunize mice. An immune response was achieved in all mice treated with H5-zein, even at low doses. The fusion to zein markedly enhanced the IgG response compared the soluble H5 control, and the effect was similar to a commercial adjuvant. The co-administration of adjuvants with the H5-zein bodies did not enhance the immune response any further, suggesting that the zein portion itself mediates an adjuvant effect. While the zein portion used to induce protein body formation was only weakly immunogenic, our results indicate that zein-induced PBs are promising production and delivery vehicles for subunit vaccines.