Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-017-0458-x) contains supplementary material, which is available to authorized users.

A Plant-Based Artificial Haemagglutinin (A/H5N1) Strongly Induced Neutralizing Immune Responses in Mice

“…The differences in cleavage sites and antigenic sites between artificial and native H5 sequences are described in yellow and green color, respectively, and all of the H5 sequences removed at the cleavage site 342-344/345 are shown in Table 1. Native HA proteins have been expressed successfully in plants [21,24]. In this study, artificial HA proteins were also expressed and detected in the crude extracts of tobacco leaves (as shown in Figure 3a).…”

“…The expression of recombinant protein by agrobacterium infiltration was based on the protocol of Phan and colleagues [23,24]. Agrobacteria containing the shuttle vectors for the expression of recombinant HcPro [25,26] and HA proteins were precultivated independently in 50 ml of LB medium containing 50 µg/mL kanamycin, 50 µg/mL carbenicillin, and 50 µg/mL rifampicin at 28 • C overnight at a speed of 140 rpm.…”

“…IMAC was based on the method described by Phan and colleagues [24], with some modifications. Eighty grams of tobacco leaf samples were ground in liquid nitrogen and blended in 240 mL of 50 mM Tris buffer (pH 8.0).…”

“…For H5.c1 protein purification, the different concentration of imidazole (30 mM, 25 mM, 20 mM, or 5 mM imidazole) in 2L of washing buffer (50 mM NaH2PO4, 300 mM NaCl) was tested Finally, H5.c1 protein was released from the column by adding 20 ml of elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 50 mM imidazole with artificial H5.c1, pH 8.0), concentrated in PEG 6000 and dialyzed against PBS. The native H5TG protein was purified successfully with 30 mM and 125 mM imidazole at the washing step and elution step, respectively [24]. Protein contents of purification steps were determined by Bradford assay [27].…”

“…The SEC was based on the method described by Phan and colleagues [24]. Equal amounts of purified artificial H5.c1 and native H5TG were introduced into a Superose™ 6 10/300GL column (GE Healthcare) which was pre-equilibrated with phosphate-buffered saline (PBS).…”