Abstract
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.
Highlights
Influenza A viruses, negative-stranded enveloped orthomyxoviruses, are among the most serious respiratory pathogens
Recombinant hemagglutinin‐S·Tag fusion proteins and S·Protein variants are produced in planta Trimeric hemagglutinin containing S·Tag and S·Protein variants were designed and expressed in planta, to generate influenza hemagglutinin oligomers based on the specific interaction of bovine S·Protein and the S·Tag (Figure 1)
All plant-made hemagglutinin ectodomains tested to date were expressed as soluble monomeric [10, 32] as well as trimeric proteins stabilized by trimeric motifs [10, 31]
Summary
Influenza A viruses, negative-stranded enveloped orthomyxoviruses, are among the most serious respiratory pathogens. They cause severe and potentially fatal illnesses [1]. Pathogenic avian flu influenza viruses are expected to cause the global pandemic threat due to their relative easy spread by avian hosts and their ability to directly infect humans [2]. During several H5N1 outbreaks, very large direct and indirect impacts on poultry and tourist industries in South-East Asia were observed [3]. Highly pathogenic avian influenza viruses (HPAI) A (H5N8) caused outbreaks in South Korea, China and Japan and were reported in many European countries as well as in the US and Canada (for an review, see [4]).
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