Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope

Abstract

Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15-30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant "universal" vaccine against influenza A produced in plants.

Keywords: M2e peptide; Nicotiana benthamiana; influenza A; nanoparticle; plant; self-assembling peptide; transient expression; vaccine; viral vector.

Figure 1

Expression vector and recombinant protein. Scheme of the expression vector (a). RDRP, RNA-dependent RNA polymerase gene; Sgp1, the first promoter of subgenomic RNA of PVX; AMV, translational enhancer from alfalfa mosaic virus; 35S, promoter of the cauliflower mosaic virus RNA; Nos-T, terminator of the A. tumefaciens nopaline synthase gene; P24, gene of suppressor of silencing from grapevine leafroll-associated virus-2. 6H, hexahistidine tag; 19S, flexible glycine-rich linker; SAP, self-assembling peptide; SP, rigid helical linker; 4M2eh, four tandem copies of M2e peptide; RB and LB, the right and left borders of T-DNA region. Models of the three-dimensional structure of monomeric 19S_SAP_Sp_4M2eh protein and nanoparticles composed of 60 monomers (b).

Figure 2

Expression of 19S_SAP_Sp_4M2eh protein in N. benthamiana plants. Coomassie brilliant blue-stained gel (a,c) and Western blotting with antibodies against M2e (b) of proteins isolated from plants. M, molecular weight marker (kD); Lanes: 1, total proteins isolated from non-infiltrated leaf; 2, total proteins isolated from leaf infiltrated with pEff/19S_SAP_Sp_4M2eh; 3, purified 19S_SAP_Sp_4M2eh protein. Position of 19S_SAP_Sp_4M2eh protein is shown by arrow. Photographs of N. benthamiana leaves infiltrated with pEff/19S_SAP_Sp_4M2eh on different dpi (d).

Figure 3

Analysis of virus-like particles formed by 19S_SAP_Sp_4M2eh protein by atomic force microscopy (a) and transmission electron microscopy (b).

Figure 4

Immunogenicity and protective efficiency of 19S_SAP_Sp_4M2eh nanoparticles. Titers of IgG antibodies to synthetic M2e peptide in sera (a) and BAL (b) of immunized mice after the third immunization. The values for 4 animals (circles and squares) and the geometric mean titers (horizontal lines) are shown. The survival rate (c) and body weight changes (d) of immunized and control (PBS) mice was monitored for 14 days post-challenge with 4 × LD₅₀ of A/Aichi/2/68 (H3N2) influenza A virus.